Antiviral vaccines with improved cellular immunogenicity

ABSTRACT

The invention provides compositions, methods, and kits for the treatment or prevention of viral infections. The polyvalent (e.g., 2-valent) vaccines described herein incorporate computationally-optimized viral polypeptides that can increase the diversity or breadth and depth of cellular immune response in vaccinated subjects.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a National Stage Application of PCT/US2009/064999,filed Nov. 18, 2009, which claims the benefit of the filing date of U.S.provisional patent application No. 61/115,703, filed Nov. 18, 2008; U.S.provisional application No. 61/152,184, filed Feb. 12, 2009; and U.S.provisional application No. 61/248,188, filed on Oct. 2, 2009, each ofwhich is hereby incorporated by reference.

STATEMENT OF FEDERALLY FUNDED RESEARCH

This research has been sponsored in part by NIH grant numbersU19-AI066305 and U19-AI078526. The government has certain rights to theinvention.

FIELD OF THE INVENTION

The invention provides compositions, methods, and kits for the treatmentor prevention of viral infections. The polyvalent (e.g., 2-valent)vaccines described herein incorporate computationally-optimized viralpolypeptides that can increase the diversity or breadth and depth ofcellular immune response in vaccinated subjects.

BACKGROUND OF THE INVENTION

Vaccines that elicit cellular immune responses against viruses mustreflect global viral diversity in order to effectively treat or preventviral infection. For example, the initiation of intense and diverseHIV-1-specific T cell responses is likely crucial for an effective HIV-1vaccine. Cytotoxic T lymphocyte (CTL) responses are correlated with slowdisease progression in humans, and the importance of CTL responses innon-human primate vaccination models is well established. While thehighly variable Envelope (Env) is the primary target for neutralizingantibodies against HIV, and vaccine antigens will also need to betailored to elicit these antibody responses, T cell vaccine componentscan target more conserved proteins to trigger responses that are morelikely to cross-react. But even the most conserved HIV-1 proteins arediverse enough that variation will be an issue. Artificialcentral-sequence vaccine approaches, such as consensus and ancestralHIV-1 sequences, essentially “split the differences” between strains,can stimulate responses with enhanced cross-reactivity compared tonatural strain vaccines. Consensus antigens represent synthetic antigensequences that are the single best “average” of all circulating strains.While these antigens can elicit directed cellular immune responses, thebreadth and intensity of these responses are not substantially improvedover previous vaccine strategies. The development of next-generationvaccines to treat or prevent viral infection must elicit an increasedbreadth of cellular immunity in order to allow for successfulvaccination outcomes. The need for such vaccines is particularly urgentfor the treatment or prevention of HIV-1.

SUMMARY OF THE INVENTION

In a first aspect, the invention features a vaccine for treating orreducing the risk of a viral infection in a mammal, such as a human,that includes at least two distinct optimized viral polypeptides (e.g.,2, 3, 4, 5, or more distinct optimized viral polypeptides), wherein theoptimized viral polypeptides correspond to the same viral gene product.In one embodiment, the viral infection is caused by a retrovirus,reovirus, picornavirus, togavirus, orthomyxovirus, paramyxovirus,calicivirus, arenavirus, flavivirus, filovirus, bunyavirus, coronavirus,astrovirus, adenovirus, papillomavirus, parvovirus, herpesvirus,hepadnavirus, poxvirus, or polyomavirus. In other embodiments, theretrovirus is human immunodeficiency virus type 1 (HIV-1), and the viralgene products include Gag, Pol, Env, Nef, Tat, Rev, Vif, Vpr, or Vpu. Ina further embodiment, the vaccine includes no more than two optimizedviral polypeptides corresponding to one of the Gag, Pol, Env, Nef, Tat,Rev, Vif, Vpr, or Vpu viral gene products. In another embodiment, thevaccine does not include optimized viral polypeptides corresponding toGag and Nef. In yet another embodiment, the vaccine includes at leasttwo distinct optimized viral polypeptides (e.g., 2, 3, 4, 5, or moredistinct optimized viral polypeptides) for a first viral gene productselected from Gag, Pol, Env, Nef, Tat, Rev, Vif, Vpr, and Vpu and one ormore distinct optimized viral polypeptides (e.g., 2, 3, 4, 5, or moredistinct optimized viral polypeptides) for a second viral gene productdifferent from the first viral gene product selected from Gag, Pol, Env,Nef, Tat, Rev, Vif, Vpr, and Vpu.

In a second aspect, the invention features a vaccine for treating orreducing the risk of human immunodeficiency virus type 1 (HIV-1)infection in a mammal, such as a human, that includes an optimized viralpolypeptide that has at least seven contiguous amino acids (e.g., atleast 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24,25, 26, 27, 28, 29, 30, 50, 100, 150, 175, 200, 250, 300, 350, 400, 450,500 or more contiguous amino acids in length) having at least 85% aminoacid sequence identity to any one of the sequences set forth in SEQ IDNOS:1-29. In one embodiment, the optimized viral polypeptide has atleast seven contiguous amino acids (e.g., at least 8, 9, 10, 11, 12, 13,14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 50,100, 150, 175, 200, 250, 300, 350, 400, 450, 500 or more contiguousamino acids in length) having amino acid sequence identity to any one ofthe sequences set forth in SEQ ID NOS:1-29. In another embodiment, theoptimized viral polypeptide has the amino acid sequence of any one ofthe sequences set forth in SEQ NOS:1-29. In a further embodiment, thevaccine includes at least two optimized viral polypeptides selected fromany one or more of groups a)-k): a) SEQ ID NOS:1 and 2; b) SEQ ID NOS:3,4, and 5; c) SEQ ID NOS:6 and 7; d) SEQ ID NOS:8-12; e) SEQ ID NOS:13,14, and 15; SEQ ID NOS:16, 17, and 18; g) SEQ ID NOS:19 and 20; h) SEQID NOS:21, 22, and 23; i) SEQ ID NOS:24 and 25; j) SEQ ID NOS:26 and 27;k) and SEQ ID NOS:21-22. In another embodiment, the vaccine can includea pair of optimized viral polypeptides selected from any one of groupsa)-k) above and one or more different optimized viral polypeptides fromthe same or a different group a)-k). In other embodiments, the vaccinecan include at least three or four or more optimized viral polypeptidesfrom one or more of groups a)-k).

In a third aspect, the invention features a vaccine for treating orreducing the risk of a viral infection in a mammal, such as a human,that includes at least two pairs of distinct optimized viralpolypeptides, wherein each pair of optimized viral polypeptidescorresponds to the same viral gene product, and wherein no more than twooptimized viral polypeptides incorporated in the vaccine correspond tothe same viral gene product. In one embodiment, the vaccine includes atleast three pairs of distinct optimized viral polypeptides. In anotherembodiment, the vaccine includes at least four pairs of distinctoptimized viral polypeptides. In one embodiment, the viral infection iscaused by a retrovirus, reovirus, picornavirus, togavirus,orthomyxovirus, paramyxovirus, calicivirus, arenavirus, flavivirus,filovirus, bunyavirus, coronavirus, astrovirus, adenovirus,papillomavirus, parvovirus, herpesvirus, hepadnavirus, poxvirus, orpolyomavirus. In other embodiments, the retrovirus is humanimmunodeficiency virus type 1 (HIV-1), and the viral gene productsinclude Gag, Pol, Env, Nef, Tat, Rev, Vif, Vpr, or Vpu. In a furtherembodiment, the vaccine includes no more than two optimized viralpolypeptides corresponding to one of the Gag, Pol, Env, Nef, Tat, Rev,Vif, Vpr, or Vpu viral gene products. In another embodiment, the vaccinedoes not include optimized viral polypeptides corresponding to Gag andNef. In a further embodiment, the vaccine includes at least three pairsof distinct optimized viral polypeptides corresponding to any three ofthe Gag, Pol, Env, Nef, Tat, Rev, Vif, Vpr, or Vpu viral gene products.In another embodiment, the vaccine includes at least four pairs ofdistinct optimized viral polypeptides corresponding to any four of theGag, Pol, Env, Nef, Tat, Rev, Vif, Vpr, or Vpu viral gene products.

In one embodiment of any of the first three aspects of the invention,the vaccine elicits a cellular immune response against a viral geneproduct. In another embodiment, the vaccine elicits a cellular immuneresponse against HIV-1. In a further embodiment, the nucleotide sequenceof at least one distinct optimized viral polypeptide is encoded by anucleic acid or vector. In one embodiment, the vector is a recombinantadenovirus, such as adenovirus serotype 26 (Ad26), adenovirus serotype34 (Ad34), adenovirus serotype 35 (Ad35), adenovirus serotype 48 (Ad48),or adenovirus serotype 5 HVR48 (Ad5HVR48). In a further embodiment, thevaccine is in combination with a pharmaceutically acceptable carrier,excipient, or diluent.

In a fourth aspect, the invention features a nucleic acid that includesthe nucleotide sequence of an optimized viral polypeptide that has atleast seven contiguous amino acids (e.g., at least 8, 9, 10, 11, 12, 13,14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 50,100, 150, 175, 200, 250, 300, 350, 400, 450, 500 or more contiguousamino acids in length) having at least 85% amino acid sequence identityto any one of the amino acid sequences set forth in SEQ ID NOS:1-29. Inone embodiment, the optimized viral polypeptide has at least sevencontiguous amino acids (e.g., at least 8, 9, 10, 11, 12, 13, 14, 15, 16,17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 50, 100, 150,175, 200, 250, 300, 350, 400, 450, 500 or more contiguous amino acids inlength) having sequence identity to any one of the amino acid sequencesset forth in SEQ ID NOS:1-29. In another embodiment, the optimized viralpolypeptide has any one of the amino acid sequences set forth in SEQ IDNOS:1-29. In a further embodiment, the nucleic acid includes a vector.In one embodiment, the vector is a recombinant adenovirus, such asadenovirus serotype 26 (Ad26), adenovirus serotype 34 (Ad34), adenovirusserotype 35 (Ad35), adenovirus serotype 48 (Ad48), or adenovirusserotype 5 HVR48 (Ad5HVR48).

In a fifth aspect, the invention features an optimized viral polypeptidethat has at least seven contiguous amino acids (e.g., at least 8, 9, 10,11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28,29, 30, 50, 100, 150, 175, 200, 250, 300, 350, 400, 450, 500 or morecontiguous amino acids in length) having at least 85% amino acidsequence identity to any one of the amino acid sequences set forth inSEQ ID NOS:1-29. In one embodiment, the optimized viral polypeptide hasat least seven contiguous amino acids (e.g., at least 8, 9, 10, 11, 12,13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30,50, 100, 150, 175, 200, 250, 300, 350, 400, 450, 500 or more contiguousamino acids in length) having sequence identity to any one of the aminoacid sequences set forth in SEQ ID NOS:1-29. In another embodiment, theoptimized viral polypeptide has any one of the amino acid sequences setforth in SEQ ID NOS:1-29.

In a sixth aspect, the invention features a method for treating orreducing the risk of a viral infection in a mammal, such as a human, byadministering a vaccine or nucleic acid of the invention. In oneembodiment, the viral infection is caused by a retrovirus, reovirus,picornavirus, togavirus, orthomyxovirus, paramyxovirus, calicivirus,arenavirus, flavivirus, filovirus, bunyavirus, coronavirus, astrovirus,adenovirus, papillomavirus, parvovirus, herpesvirus, hepadnavirus,poxvirus, or polyomavirus. In further embodiments, the retrovirus ishuman immunodeficiency virus type 1 (HIV-1), and the viral gene productsinclude Gag, Pol, Env, Nef, Tat, Rev, Vif, Vpr, or Vpu. In oneembodiment, the vaccine or nucleic acid elicits a cellular immuneresponse against a viral gene product.

In a seventh aspect, the invention features a method of manufacturing avaccine for treating or reducing the risk of a viral infection in amammal, such as a human, by synthesizing a vaccine of the invention.

In an eighth aspect, the invention features a method of manufacturing avaccine for treating or reducing the risk of a viral infection in amammal, such as a human, by contacting a nucleic acid of the inventionwith a cell and isolating a optimized viral polypeptide.

In one embodiment of the seventh or eighth aspects of the invention, theoptimized viral polypeptide elicits a cellular immune response whenadministered to a mammal. The cellular immune response can be against aviral gene product. In another embodiment, the viral infection is causedby a retrovirus, reovirus, picornavirus, togavirus, orthomyxovirus,paramyxovirus, calicivirus, arenavirus, flavivirus, filovirus,bunyavirus, coronavirus, astrovirus, adenovirus, papillomavirus,parvovirus, herpesvirus, hepadnavirus, poxvirus, or polyomavirus. Infurther embodiments, the retrovirus is human immunodeficiency virus type1 (HIV-1), and the viral gene products include Gag, Pol, Env, Nef, Tat,Rev, Vif, Vpr, or Vpu.

In a ninth aspect, the invention features a kit that includes a vaccineof the invention, a pharmaceutically acceptable carrier, excipient, ordiluent, and instructions for the use thereof. In one embodiment, thekit also includes an adjuvant.

In a final aspect, the invention features a kit that includes a nucleicacid of the invention, a pharmaceutically acceptable carrier, excipient,or diluent, and instructions for the use thereof. In one embodiment, thekit also includes an adjuvant.

In an embodiment of all aspects of the invention, the optimized viralpolypeptide is encoded by a nucleic acid sequence that is optimized forexpression in humans (e.g., any one of SEQ ID NOS:5, 10, 11, 12, 15, 18,and 23).

DEFINITIONS

By “optimized viral polypeptide” or “computationally-optimized viralpolypeptide” is meant an immunogenic polypeptide that is not anaturally-occurring viral peptide, polypeptide, or protein. Optimizedviral polypeptide sequences are initially generated by modifying theamino acid sequence of one or more naturally-occurring viral geneproducts (e.g., peptides, polypeptides, and proteins) to increase thebreadth, intensity, depth, or longevity of the antiviral immune response(e.g., cellular or humoral immune responses) generated upon immunization(e.g., when incorporated into a vaccine of the invention) of a mammal(e.g., a human). Thus, the optimized viral polypeptide may correspond toa “parent” viral gene sequence; alternatively, the optimized viralpolypeptide may not correspond to a specific “parent” viral genesequence but may correspond to analogous sequences from various strainsor quasispecies of a virus. Modifications to the viral gene sequencethat can be included in an optimized viral polypeptide include aminoacid additions, substitutions, and deletions. In one embodiment of theinvention, the optimized viral polypeptide is the composite or mergedamino acid sequence of two or more naturally-occurring viral geneproducts (e.g., natural or clinical viral isolates) in which eachpotential epitope (e.g., each contiguous or overlapping amino acidsequence of 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20,21, 22, 23, 24, 25, 26, 27, 28, 29, or more amino acids in length) isanalyzed and modified to improve the immunogenicity of the resultingoptimized viral polypeptide. Optimized viral polypeptides thatcorrespond to different viral gene products can also be fused tofacilitate incorporation in a vaccine of the invention. Methods ofgenerating an optimized viral polypeptides are described in, e.g.,Fisher et al. “Polyvalent Vaccine for Optimal Coverage of PotentialT-Cell Epitopes in Global HIV-1 Variants,” Nat. Med. 13(1):100-106(2007) and International Patent Application Publication WO 2007/024941,herein incorporated by reference. Once the optimized viral polypeptidesequence is generated, the corresponding polypeptide can be produced oradministered by standard techniques (e.g., recombinant viral vectors,such as the adenoviral vectors disclosed in International PatentApplication Publications WO 2006/040330 and WO 2007/104792, hereinincorporated by reference).

By “pharmaceutically acceptable carrier” is meant a carrier which isphysiologically acceptable to the treated mammal while retaining thetherapeutic properties of the compound with which it is administered.One exemplary pharmaceutically acceptable carrier is physiologicalsaline. Other physiologically acceptable carriers and their formulationsare known to one skilled in the art and described, e.g., in Remington'sPharmaceutical Sciences (18^(th) edition, ed. A. Gennaro, 1990, MackPublishing Company, Easton, Pa.), incorporated herein by reference.

By “vector” is meant a DNA construct that contains a promoter operablylinked to a downstream gene or coding region (e.g., a cDNA or genomicDNA fragment, which encodes a polypeptide or polypeptide fragment).Introduction of the vector into a recipient cell (e.g., a prokaryotic oreukaryotic cell, e.g., a bacterium, yeast, insect cell, or mammaliancell, depending upon the promoter within the expression vector) ororganism (including, e.g., a human) allows the cell to express mRNAencoded by the vector, which is then translated into the encodedoptimized viral polypeptide of the invention. Vectors for in vitrotranscription/translation are also well known in the art and aredescribed further herein. A vector may be a genetically engineeredplasmid, virus, or artificial chromosome derived from, e.g., abacteriophage, adenovirus, retrovirus, poxvirus, or herpesvirus.

By “viral gene product” is meant any naturally-occurring viral peptide,polypeptide, or protein, or fragment thereof. In one embodiment of theinvention, the viral gene product is derived from the humanimmunodeficiency virus type 1 (HIV-1). HIV-1 viral gene products includethe Gag, Pol, Env, Nef, Tat, Rev, Vif, Vpr, and Vpu polypeptides.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a chart that illustrates the expanded breadth ofcomputationally-optimized HIV-1 Gag, Pol, and Env viral polypeptidesagainst global potential T-cell epitopes (PTE) peptides in Rhesusmacaques. Animals immunized with the optimized viral polypeptides (blue)reacted with the greatest number of recall peptide pools.

FIG. 2 is a chart that shows that computationally-modified HIV-1 Gag,Pol, and Env viral polypeptides expand the breadth of epitope-specificcellular immune response.

FIG. 3 illustrates the breadth of cellular immune responses detected inRhesus macaques following immunization with HIV-1 viral gene productsGag, Pol, and Env derived from the computationally-modified viralpolypeptides of the invention, as well as animals immunized withconsensus HIV-1 antigens or HIV-1 clade C isolate antigens. Animalsimmunized with the optimized viral polypeptides (blue) reacted with thegreatest number of recall peptide pools. Since the animals are outbred,the pools differ from animal to animal. Gag, Pol, and Env each elicitmany cellular immune responses and can have shared patterns ofreactivity.

FIGS. 4A-4C are graphs showing the potential epitopes shared between thedifferent vaccines tested (2 valent mosaic (Mos2), M consensus (Mcon),and optimized clade C (OptC)) by viral polypeptide (Pol (FIG. 4A), Gag(FIG. 4B), and Env (FIG. 4C)). FIGS. 4A-C show the relative coverage ofthe current HIV database full length genome set and the PTE peptides bythe different vaccine candidates.

FIG. 5 is a graph showing that the number of PTE peptide responses(where each response is considered an independent event regardless ofoverlap) to the 2 valent mosaic (Mos2) vaccine is greater than thenumber of responses to the M group consensus (Mcon) vaccine and thenatural viral strain vaccine (optimized clade C(C Natural (optimal)),which has been selected to give optimal coverage of the M groupcollection (OptC) vaccine antigens. FIG. 5 shows the number of PTEpeptide responses per animal by protein, CD8+ T cell, and CD4+ T cell.Statistically, Mos2>Mcon˜OptC (Mcon shows a trend for more response thanOptC). The Wilcoxon p-value for Mos2 compared to Mcon: p-value=0.001058.

FIG. 6 is a chart showing the number of PTE peptides that trigger T cellresponses. A median number of 16 (range; 12-29) PTE peptides of the 2valent mosaic (Mos2) vaccine trigger a response in CD8+ T cells, whileonly a median number of 6 (range: 0-7) Mcon peptides and only a mediannumber of 3 peptides (range: 0-3) of OptC peptides trigger a response inCD8+ T cells. A median number of 4 (range; 2-6) PTE peptides of the 2valent mosaic (Mos2) vaccine trigger a response in CD4+ T cells, whileonly a median number of 1 (range: 0-2) Mcon peptides and only a mediannumber of 0.5 peptides (range: 0-2) of OptC peptides trigger a responsein CD4+ T cells. Thus, the trend for responses is Mos2>Mcon>OptC.

FIG. 7 is a schematic summarizing the mapping of all CD8+ T cell Gag PTEpeptides that are recognized by T cells from each of the animals studied(see Example 3 below). The animal number, peptide pool and peptidenumber label the boundaries of each reactive peptide. The symbolsignifies the group: *, Mos2; ¥, ConM; ±, OptC. Gag is included here asan example. There tends to be clustering of CD8 responses even thoughthe animals are outbred. Mosaics have potential advantages over themonovalent vaccines. Mosaics have a better chance of stimulating aresponse that reacts with more common variants. Mosaics also stimulatemultiple responses to the different forms that are present in thecocktail. Thus, mosaics have the potential to block common escaperoutes. In our study, the mosaic vaccine tended to stimulate T cellresponses that recognized more overlapping peptides. There are manyhotspots of localization of reactive peptides. PTE peptides are designedto maximize the potential epitope (or 9-mer for a 9 amino acidcontiguous stretch) coverage of the HIV-1 M group in the peptidereagents used to assess vaccines. Inevitably, there is a lot of overlapin PTE peptides, but because of the algorithm, overlap is usually anoverlap with some variation. FIG. 7 discloses SEQ ID NO: 42.

FIG. 8 is a schematic summarizing the mapping of CD4+ T cell Gag PTEpeptides that are recognized by T cells from each of the animalsstudied. FIG. 8 discloses SEQ ID NO: 43.

FIG. 9 is a chart illustrating typical patterns of PTE responses to theConM vaccine or to the optimal natural vaccine, aligning peptides thatelicit a response with the relevant region of the vaccine. Good matcheswith solid stretches of identity between vaccine and target PTE peptideare necessary to achieve a reaction to these vaccines. FIG. 9 disclosesSEQ ID NOS 44-57, respectively, in order of appearance.

FIG. 10 is a chart illustrating that mosaic vaccines generated manyresponses that recognized multiple variant overlapping peptides with noapparent antigenic competition and with broad local responses. Inparticular, four variable PTE peptides were recognized. Moreover, in theregion of overlap both mosaic forms were recognized, as well acombination of the two. Finally, a new form (S) was recognized. FIG. 10discloses SEQ ID NOS 58-63, respectively, in order of appearance.

FIG. 11 is a chart illustrating a typical pattern of CD8+ PTE peptideresponses in mosaic vaccinated animal (361-07). 22 PTE peptides weretested and 8 CD8 responsive regions were identified; 5 regions includedvariable peptides that match amino acids in one or the other of themosaics. 5 CD4 responsive regions were identified. Thus, T cellresponses to Mosaics see more variable peptides in a given region. Thisseemed to be true of CD8 T cell responses in particular. This could bethe result of triggering multiple T cell clones that recognize variantsof epitopes, and these may block fit escape routes. Not only are theremore responses, they are deeper and cover more variants. FIG. 11discloses CD8 responses as SEQ ID NOS 64-101, respectively, in order ofappearance. CD4 responses disclosed as SEQ ID NOS 102-117, respectively,in order of appearance.

FIG. 12 is a graph showing the number of overlapping variable PTEpeptides that span regions targeted by vaccine elicited T cells.

FIG. 13 is graph showing that the 2 mosaic antigen vaccine yields more Tcell responses, relative to the Mcon and OptC vaccines, to regions thatcontain one or more overlapping PTE peptides. FIG. 13 is similar to FIG.5, monkeys shown in the same order from right to left, but with thescale changed to reflect number of responses to regions that contain oneor more overlapping PTE peptides rather than single peptides.

FIG. 14 is a chart showing the number of T cell responses in animalsfollowing administration of 2 valent mosaic (Mos2), Mcon, and OptCvaccines. The 2 valent mosaic (Mos2) vaccine triggers a median number of8 responses in CD8+ T cells, while only a median number of 3 (range:0-6) and 1.5 peptides (range: 0-5) CD8+ T cell responses are triggeredby Mcon and OptC vaccines, respectively. The 2 valent mosaic (Mos2)vaccine triggers a median number of 3 (range; 2-5) responses in CD4+ Tcells, while only a median number of 1 (range: 0-2) and 0.5 (range: 0-2)CD4+ T cell response are triggered by Mcon and OptC vaccines,respectively. Thus, the trend for responses is Mos2>Mcon>OptC.

FIG. 15 is a graph showing that the mosaic vaccines can elicit moreresponses that cross-react with C clade natural proteins than can a Cclade natural vaccine: GAG pooled peptides representing 5 proteins.Animals vaccinated with the M group consensus or the optimal coverage Cclade natural protein had 0-2 responses to the peptides derived fromthese proteins, while the Mosaic vaccinated animals could respond to 1-5peptide pools. The Mosaic vaccine elicits more responses to each of theproteins tested than either M con or the optimal C. T cell responseselicited by mosaic vaccines also recognized more pooled peptide setsspanning actual Gag proteins. 10-12 Subpools=10×15mer peptides (except96ZM Gag, which is 5×20mer peptides).

FIG. 16 is a graph showing that the mosaic design is robust to changesin viral polypeptides over time (e.g., Gag M).

FIG. 17 is a graph showing that coverage using 9-mer optimization isrobust over near (e.g., 8-12 mers) optimization lengths (Gag is shown).

FIG. 18 is a graph showing that an increase in the number of variantsincreases coverage, but has only diminishing returns (Gag is shown).

FIGS. 19A-19B are graphs showing the breadth and magnitude ofepitope-specific T lymphocyte responses to PTE peptides. FIG. 19A is agraph showing the numbers of epitope-specific CD4+ (top) and CD8+(bottom) T lymphocyte responses to individual PTE peptides following asingle immunization of rAd26 vectors expressing mosaic (blue), Mconsensus (green), clade B+clade C (purple), or optimal natural clade C(red) HIV-1 Gag, Pol, and Env antigens. Individual monkeys are depictedon the x-axis. The different shades of each color reflect responses tothe different antigens (Gag, Pol, Env). FIG. 19B is a graph showing thenumbers of CD4+ (top) and CD8+ (bottom) T lymphocyte response regions.

FIGS. 20A-20C show a schematic showing CD8+ T lymphocyte responses toPTE peptides at week 4 following immunization mapped on HIV-1 Gag (FIG.20A) (SEQ ID NO: 118), Pol (FIG. 20B) (SEQ ID NO: 119), and Env (FIG.20C) (SEQ ID NO: 120) protein sequences. Colors denote monkeys thatreceived the mosaic (blue), M consensus (green), clade B+clade C(purple), or optimal natural clade C (red) HIV-1 Gag, Pol, and Envantigens. For each epitope, the monkey number, antigen (G, Gag; P, Pol;E, Env), subpool number, and individual PTE peptide number areindicated.

FIGS. 21A-21C show a schematic showing CD4+ T lymphocyte responses toPTE peptides at week 4 following immunization mapped on HIV-1 Gag (FIG.21A) (SEQ ID NO: 121), Pol (FIG. 21B) (SEQ ID NO: 122), and Env (FIG.21C) (SEQ ID NO: 123) protein sequences. Colors denote monkeys thatreceived the mosaic (blue), M consensus (green), clade B+clade C(purple), or optimal natural clade C (red) HIV-1 Gag, Pol, and Envantigens. For each epitope, the monkey number, antigen (G, Gag; P, Pol;E, Env), subpool number, and individual PTE peptide number areindicated.

FIG. 22 is a schematic showing the alignment of vaccine sequences withreactive PTE peptides in all monkeys at week 4 following immunizationwith rAd26 vectors expressing mosaic, M consensus, clade B+clade C, oroptimal natural Blade C HIV-1 Gag, Pol, and Env antigens. For eachmonkey, vaccine sequences are shown on the top, and reactive PTEpeptides are shown beneath the vaccine sequences denoted by the antigen(G, Gag; P, Pol; E, Env) and PTE peptide numbers. The minimal overlapregion is shown in bold. Sequence polymorphisms between the two mosaicor the two clade B+clade C antigens are shown in blue. Differencesbetween the vaccine sequences and the reactive PTE peptides are shown inred. FIG. 22 discloses SEQ ID NOS124-640, respectively, in order ofappearance.

Minimal regions within the peptides that are likely to contain theimmune response epitope, based on overlap between reactive peptides whenit occurs, are in bold in the vaccines. If there is no overlappingpeptide, we assume the epitope can be anywhere in the peptide, so thewhole region is bold. We cannot differentiate between different T cellresponses targeting epitopes with different boundaries within a peptide,or more promiscuous clonal T cell responses that can tolerate variationwhen variants are present; either scenario could be advantageous in avaccine immune response. The number of targeted regions

corresponds to the minimum number of T cell responses required toaccount for the data.

Amino acids where the vaccine and the peptides don't match are writtenin red; if they fall within the region likely to carry the epitope, theyare bold red. Amino acid differences outside of the overlapping regionswhen multiple peptides overlap are marked in red, but not bold.

The vaccines are always at the top. The letter for each protein (Gag isG, Pol is P, Envelope is E) and the peptide number are used to label foreach reactive PTE peptide. The protein and HXB2 numbers follow eachpeptide.

For the mosaic and clade B+C vaccines, there are 2 antigens each andboth are included in the alignment; amino acid differences in thevaccines are noted in blue, and if the reactive peptide carries thevariant amino acid in the second mosaic, it is also in blue. In each ofthe positions where the two vaccine antigens differ, the reactivepeptides are also marked in bold to indicate the positions whereincluding two variants may have impacted the vaccine immune response andallowed greater breadth and depth.

For example, the first vaccine summarized is the clade B+C vaccine, andanimal 287-95 is the first animal for which responses are listed. Therewere 3 CD8 responses to PTE peptides, 1 to CD4. Two of the CD8 peptidesshow substantial overlap, E26 and E282, so both may be targets for thesame CTL response; thus we also note there are only 2 CD8 responsiveregions, and 1 CD4 responsive region. For each responsive region, wewrite out the number of overlapping peptides per region (e.g., CD8:12CD4:1) to assess depth of responses; the two is red to indicate thatthe region of overlap is variable in the reactive peptides. If thevaccine differs, like the D/E in the second reactive region, it ismarked in blue. Only the region of overlap is bold. The H in E282 wasnot found in either vaccine so it is marked with red; it is within theregion of overlap so it is bold. Each reactive peptide has its proteinand corresponding HXB2 numbering noted on the right.

FIGS. 23A-23C are graphs showing the magnitude of all Gag-, Pol-, andEnv-specific CD8+ (FIGS. 23A and 23B) and CD4+ (FIG. 23C) T lymphocyteresponses arranged from lowest to highest.

FIGS. 24A-C show the depth of epitope-specific T lymphocyte responses toPTE peptides. FIG. 24A is a schematic showing an example of mapped Tlymphocyte responses in monkey 366 that received the optimal naturalclade C antigens. FIG. 24B is a schematic showing an example of mapped Tlymphocyte responses in monkey 361 that received the 2-valent mosaicantigens. In FIGS. 24A and 24B, vaccine sequences are shown on the top(OptC; Mos1, Mos2), and reactive PTE peptides are shown beneath thevaccine sequences denoted by the antigen (G, Gag; P, Pol; E, Env) andthe PTE peptide numbers. The minimal overlap region is shown in bold.Sequence polymorphisms between the two mosaic antigens are shown inblue. Differences between the vaccine sequences and the reactive PTEpeptides are shown in red. Complete alignments of all positive peptidesorganized by response regions are shown in FIG. 22. FIG. 24C is a graphshowing the depth of CD4+ (top) and CD8+ (bottom) T lymphocyte responsesfollowing immunization with rAd26 vectors expressing mosaic, Mconsensus, clade B+clade C, or optimal natural clade C antigens.Individual monkeys are depicted on the x-axis. One response variant(light shade) or >1 response variants (dark shade) are shown for eachepitopic region. FIG. 24A discloses SEQ ID NOS 641-650, respectively, inorder of appearance. FIG. 24B discloses SEQ ID NOS 651-685, respective),in order of appearance.

FIG. 25 is a graph showing the breadth of epitope-specific T lymphocyteresponses to HIV-1 Gag peptides from clades A, B, and C. Breadth ofcellular immune responses was assessed utilizing subpools of overlappingpeptides from the following strains of HIV-1 Gag: clade C DU422, clade CZM651, consensus C, consensus A, and consensus B. Numbers of positivesubpools are shown following a single immunization of rAd26 vectorsexpressing mosaic (blue), M consensus (green), clade B+clade C (purple),or optimal natural clade C (red) HIV-1 Gag, Pol, and Env antigens.Individual monkeys are depicted on the x-axis.

FIG. 26A-D are graphs showing the cellular and humoral immune responsesfollowing the boost immunization. Shown are the magnitude (FIG. 26A) andbreadth (FIG. 26B) of individual T lymphocyte responses at week 4post-prime (left side of each panel) and at week 44 post-boost (rightside of each panel) for each monkey. Monkeys were primed at week 0 withrAd26 vectors and were boosted at week 40 with rAd5HVR48 vectorsexpressing mosaic, M consensus, or optimal natural clade C HIV-1 Gag,Pol, and Env antigens. Individual monkeys are depicted on the x axis. InFIG. 26A, red denotes CD8+ T lymphocyte responses, blue denotes CD4+ Tlymphocyte responses, lines depict responses observed at bothtimepoints, and dots depict responses observed at only one timepoint. InFIG. 26B, different shades of each color reflect responses to thedifferent antigens (Gag, Pol, Env), FIG. 26C is a graph showing theEnv-specific ELISA endpoint titers at weeks 0, 4, and 44. FIG. 26D is agraph showing the neutralizing antibody (NAb) titers to the tier 1 cladeA (DJ263.8), clade B (SF162.LS), and clade C (MW965.26) viruses at week44. NAb titers to murine leukemia virus as a negative control were <20for all samples.

FIG. 27 is a graph showing the theoretical coverage of PTE peptides bythe various vaccine antigens. Percentage of 9 amino acid PTE peptidesthat are covered by the mosaic (blue), M consensus (green), cladeB+clade C (purple), or optimal natural clade C (red) HIV-1 Gag, Pol, andEnv antigens are shown.

DETAILED DESCRIPTION OF THE INVENTION

The invention features optimized viral polypeptides that arecomputationally derived from naturally-occurring viral gene products.The optimized viral polypeptides of the invention allow for an increasedbreadth and depth of virus-specific immunity (e.g., cellular immunity,such as T cell-based immune responses) following immunization of asubject (e.g., a human) with one or more optimized viral polypeptides ofthe invention or vaccines (e.g., a vector) that incorporate one or moreoptimized viral polypeptides of the invention. The invention providesvaccines that can be administered to a subject (e.g., a human) infectedwith or at risk of becoming infected with a viral infection. Thevaccines of the invention incorporate at least two distinct optimizedviral polypeptides for each corresponding viral gene productrepresented. The incorporation of at least two distinct optimized viralpolypeptides allows for increased coverage and representation ofimmunogenic epitopes in the vaccine, which the inventors have foundresults in an increase in the total number of virus-specific immuneresponses following vaccination of a subject. The present invention alsoprovides methods of administering and manufacturing vaccines, vectors,and optimized viral polypeptides that to a subject (e.g., a human). Thecompositions, methods, and kits described herein can substantiallyincrease the diversity, breadth, and/or depth of the virus-specificcellular immune responses by providing at least two distinct optimizedviral polypeptides.

Optimized Viral Polypeptides of the Invention

The present invention provides for polyvalent (e.g., 2-valent) vaccinesthat incorporate computationally-optimized viral polypeptides thatcorrespond to and are derived from viral gene products that naturallycirculate. Polyvalent mosaic proteins are assembled from naturalsequences by in silico recombination and optimized to provide maximalcoverage of potential T cell epitopes (PTEs) for a given valency. Mosaicantigens are full-length proteins that are designed to preserve naturalantigen expression and processing.

The inventors have discovered that immunization with two distinctoptimized viral polypeptides corresponding to and derived from a singleviral gene product (i.e., a 2-valent vaccine) elicits a substantiallyhigher number of cellular immune responses (e.g., T cell responses) thanconventional monovalent or polyvalent vaccines that incorporatenaturally-occurring polypeptides derived from the same viral geneproduct (e.g., sequences based on clinical isolates), or a consensussequence of such naturally-occurring polypeptides derived from the sameviral gene product. Accordingly, a vaccine that incorporatescomputationally-optimized viral polypeptides, the sequences of whichprovide maximum coverage of non-rare short stretches of circulatingviral sequences, can increase the breadth and depth of the immuneresponse.

A genetic algorithm is used to create sets of optimized viralpolypeptides as “mosaic” blends of fragments of an arbitrary set ofnaturally-occurring viral gene product sequences provided as inputs.This genetic algorithm strategy uses unaligned protein sequences from ageneral viral population as an input data set, and thus has the virtueof being “alignment independent.” It creates artificial optimized viralpolypeptides that resemble viral proteins found in nature, but are notnaturally-occurring. The genetic algorithm can be adjusted to optimizeviral polypeptides of different lengths, depending on the intendedtarget or desired immune response. As most T cell epitopes are nineamino acids in length, the genetic algorithm utilized to design theoptimized viral polypeptides of the invention was based on optimizingeach consecutive 9-mer amino acid sequence of a given viral gene product(e.g., HIV-1 Gag). In accordance with this approach, 9-mers (forexample) that do not exist in nature or that are very rare can beexcluded—this is an improvement relative to consensus sequence-basedvaccine strategies since the latter can contain some 9-mers (forexample) that occur rarely or not at all in nature. The definition offitness used for the genetic algorithm is that the most “fit” polyvalentcocktail is the combination of input viral sequences that gives the bestcoverage (highest fraction of perfect matches) of all of the 9 mers inthe population and is subject to the constraint that no 9 mer is absentor rare in the population. The genetic algorithm used to generate theoptimized viral polypeptides of the invention is further described inInternational Patent Application Publication WO 2007/024941, hereinincorporated by reference.

In one embodiment, the invention provides polyvalent (e.g., 2-valent)HIV-1 vaccines that incorporate single optimized HIV-1 polypeptides(e.g., the polypeptides set forth in SEQ ID NOS:1-29). In anotherembodiment, the invention features a polyvalent vaccine thatincorporates two or more optimized HIV-1 polypeptides. In each case, theoptimized HIV-1 polypeptides are based on all HIV-1 variants in globalcirculation, known as the HIV-1 Main (M) group. The inventors havegenerated a set of optimized HIV-1 polypeptides (SEQ ID NOS:1-29) thataugment the breadth and depth of cellular immunity based on group Mmosaic genes that utilize only two variants per gene (e.g., twopolypeptide sequences each for Gag, Pol, Env, Nef, Tat, Rev, Vif, Vpr,and Vpu). We have obtained the novel and surprising result in Rhesusmacaques that the use of these optimized HIV-1 polypeptides in apolyvalent (e.g., 2-valent) HIV-1 group M vaccine elicits asignificantly greater breadth and depth of HIV-1-specific cellularimmune responses when compared with two other leading vaccine antigenstrategies (M consensus antigens and optimal natural clade C antigens).

The invention provides for the fusion of optimized viral polypeptidesthat correspond to different viral gene products. The genetic algorithmdescribed above can be used to generate fused polypeptides for use in avaccine of the invention. For example, the optimized HIV-1 polypeptidefusions of Gag/Nef (SEQ ID NOS:19-20), Gag/Pol (SEQ ID NOS:21-27), andGag/Pol/Nef (SEQ ID NOS:28-29) can be incorporated into a vector of theinvention for administration to a subject (e.g., a human) infected withor at risk of being infected with HIV-1. The vaccines of the invention(whether in polypeptide or nucleic acid form) can also include one ormore of the non-“mosaic” polypeptides (or sequences encoding them,respectively), such as, e.g., the optimal clade C sequences (SEQ ID NOS:30-36) or the consensus sequences (SEQ ID NOS: 37-39).

The optimized viral polypeptides disclosed in this invention can beprepared conventionally by chemical synthesis techniques, such asdescribed by Merrifield, J. Amer. Chem. Soc. 85:2149 (1963) (see also,e.g., Stemmer et al., 164 Gene 49 (1995)). For example, the vaccines canbe readily prepared using solid phase peptide synthesis (SPPS).Automated solid phase synthesis can be performed using any one of anumber of well known, commercially available automated synthesizers,such as the Applied Biosystems ABI 433A peptide synthesizer.Alternatively, the optimized viral polypeptides of the invention can berecombinantly produced by transfecting or transducing a cell or organismwith a nucleic acid or vector (e.g., a viral vector, such an adenovirus)that allows for the intracellular expression of the optimized viralpolypeptide. Nucleic acids and vectors that encode the nucleotidesequence of optimized viral polypeptides of the invention can besynthesized by well-known recombinant DNA techniques, including thosedescribed herein.

Vaccines of the Invention

The invention also features vaccines that can be administered to apatient infected with or at risk of becoming infected with a virus(e.g., HIV-1). A vaccine of the invention contains at least one of theoptimized viral polypeptides of the invention, as discussed herein. Thevaccine of the invention can be a nucleic acid encoding the nucleotidesequence of two or more optimized viral polypeptides of the invention(e.g., the immunogenic component of a recombinant (e.g., subunit) orwhole-organism (e.g., whole-virus) viral vector). Nucleic acids includevectors (e.g., viral vectors, such as adenoviruses) that incorporate thenucleotide sequence of two or more optimized viral polypeptides of theinvention. The optimized viral polypeptides of the invention, as well asvaccines, nucleic acids, and vectors that incorporate optimized viralpolypeptides, can be recombinantly expressed in a cell or organism, orcan be directly administered to subject (e.g., a human) infected with,or at risk of becoming infected with, a virus.

Vectors of the Invention

The invention also features vectors encoding the nucleotide sequences(e.g., DNA or RNA) of one or more optimized viral polypeptides of theinvention. The vector can be a carrier (e.g., a liposome), a plasmid, acosmid, a yeast artificial chromosome, or a virus that includes anucleotide sequence encoding one or more optimized viral polypeptides ofthe invention. The vector can include additional nucleic acid sequencesfrom several sources.

Vectors encoding one or more optimized viral polypeptides of theinvention can be constructed using any recombinant molecular biologytechnique known in the art. The vector, upon transfection ortransduction of a target cell or organism, can be extrachromosomal or itcan be integrated into the host cell chromosome. The nucleic acidcomponent of a vector can be in single or multiple copy number pertarget cell, and can be linear, circular, or concatamerized.

Vectors of the invention can also include internal ribosome entry site(IRES) sequences to allow for the expression of multiple peptide orpolypeptide chains from a single nucleic acid transcript. For example, avector of the invention can encode one or more optimized viralpolypeptides of the invention as well as another polypeptides (e.g., adetectable label, such as green fluorescent protein (GFP)).

Vectors of the invention further include gene expression elements thatfacilitate the expression of optimized viral polypeptides of theinvention. Gene expression elements useful for the expression of anvector encoding an optimized viral polypeptide of the invention include,but are not limited to (a) regulatory sequences, such as viraltranscription promoters and their enhancer elements, such as the SV40early promoter, Rous sarcoma virus LTR, and Moloney murine leukemiavirus LTR; (b) splice regions and polyadenylation sites such as thosederived from the SV40 late region; and (c) polyadenylation sites such asin SV40. Also included are plasmid origins of replication, antibioticresistance or selection genes, multiple cloning sites (e.g., restrictionenzyme cleavage loci), and other viral gene sequences (e.g., sequencesencoding viral structural, functional, or regulatory elements, such asthe HIV long terminal repeat (LTR)).

Vectors of the invention can also include optimized viral polypeptidesof the invention that have been optimized for expression in humans, suchas, e.g., any one of SEQ ID NOS:11, 14-18, and 23.

Vectors of the invention can also be engineered to include a multiplecloning site (MCS) having the following enzyme cleavage sites:XbaI-EcoRI-Kozak-Start . . . Stop-BamHI-NheI; and the followingsequence: TCTAGA GAATTC GCCACC [ATG gene TAA TGA] GGATCC GCTAGC. Vectorshaving this MCS can be used with optimized viral polypeptides having nointernal XbaI, EcoRI, BamHI, NheI sites and no stretches of 6 or moreC's or G's.

In Vivo Administration

The invention features methods for the in vivo administration of one ormore vaccines of the invention (e.g., a vector encoding two or moreoptimized viral polypeptides of the invention) to a subject (e.g., ahuman) to facilitate the expression of two or more optimized viralpolypeptides of the invention. Upon administering the vaccine to thesubject, one or more optimized viral polypeptides of the invention willbe expressed that can elicit protective or therapeutic immune responses(e.g., cellular or humoral immune responses) directed against the viralimmunogens.

Several types of vectors can be employed to deliver a nucleotidesequence encoding one or more optimized viral polypeptides of theinvention directly to a subject (e.g., a human). Vectors of theinvention include viruses, naked DNA, oligonucleotides, cationic lipids(e.g., liposomes), cationic polymers (e.g., polysomes), virosomes, anddendrimers. The present invention provides for the ex vivo transfectionor transduction of cells (e.g., blood cells) followed by administrationof these cells back into the donor subject to allow for the expressionof optimized viral polypeptides of the invention that have immunogenicproperties. Cells that can be isolated and transfected or transduced exvivo according to the methods of invention include, but are not limitedto, blood cells, skin cells, fibroblasts, endothelial cells, skeletalmuscle cells, hepatocytes, prostate epithelial cells, and vascularendothelial cells. Stem cells are also appropriate cells fortransduction or transfection with a vector of the invention. Totipotent,pluripotent, multipotent, or unipotent stem cells, including bone marrowprogenitor cells and hematopoietic stem cells (HSC), can be isolated andtransfected or transduced with an vector encoding one or more optimizedviral polypeptides of the invention, and administered to a subjectaccording to the methods of the invention.

The method of transfection or transduction used to express an optimizedviral vector of the invention has a strong influence on the strength andlongevity of protein expression in the transfected or transduced cell,and subsequently, in the subject receiving the cell. The presentinvention provides vectors that are temporal (e.g., adenoviral vectors)or long-lived (e.g., retroviral vectors) in nature. Regulatory sequences(e.g., promoters and enhancers) are known in the art that can be used toregulate protein expression. The type of cell being transfected ortransduced also has a strong bearing on the strength and longevity ofprotein expression. For example, cell types with high rates of turnovercan be expected to have shorter periods of protein expression.

Ex Vivo Transfection and Transduction

The invention also features methods for the ex vivo transfection andtransduction of cells (e.g., blood cells, such as lymphocytes), followedby administration of these cells to a subject (e.g., a human). In oneembodiment, the cells are autologous to the treated subject. Cells canbe transfected or transduced ex vivo with one or more vectors encodingthe nucleotide sequence of one or more optimized viral polypeptides ofthe invention to allow for the temporal or permanent expression of theoptimized viral polypeptides in the treated subject. Upon administeringthese modified cells to the subject, one or more optimized viral vectorsof the invention will be expressed that can elicit protective ortherapeutic immune responses (e.g., cellular or humoral immuneresponses) directed against the viral immunogens.

Several types of vectors can be employed to deliver a nucleotidesequence encoding one or more optimized viral polypeptides of theinvention to a cell (e.g., a blood cell, such as a lymphocyte). Vectorsof the invention include viruses, naked DNA, oligonucleotides, cationiclipids (e.g., liposomes), cationic polymers (e.g., polysomes),virosomes, and dendrimers. The present invention provides for the exvivo transfection or transduction of cells (e.g., blood cells) followedby administration of these cells back into the donor subject to allowfor the expression of optimized viral polypeptides of the invention thathave immunogenic properties. Cells that can be isolated and transfectedor transduced ex vivo according to the methods of invention include, butare not limited to, blood cells, skin cells, fibroblasts, endothelialcells, skeletal muscle cells, hepatocytes, prostate epithelial cells,and vascular endothelial cells. Stem cells are also appropriate cellsfor transduction or transfection with a vector of the invention.Totipotent, pluripotent, multipotent, or unipotent stem cells, includingbone marrow progenitor cells and hematopoietic stem cells (HSC), can beisolated and transfected or transduced with an vector encoding one ormore optimized viral polypeptides of the invention, and administered toa subject according to the methods of the invention.

The method of transfection or transduction used to express an optimizedviral vector of the invention has a strong influence on the strength andlongevity of protein expression in the transfected or transduced cell,and subsequently, in the subject receiving the cell. The presentinvention provides vectors that are temporal (e.g., adenoviral vectors)or long-lived (e.g., retroviral vectors) in nature. Regulatory sequences(e.g., promoters and enhancers) are known in the art that can be used toregulate protein expression. The type of cell being transfected ortransduced also has a strong bearing on the strength and longevity ofprotein expression. For example, cell types with high rates of turnovercan be expected to have shorter periods of protein expression.

Viral Vectors

Viral vectors encoding the nucleotide sequence of one or more optimizedviral polypeptides of the invention can be used as a vaccine of theinvention. For example, the nucleotide sequence of one or more optimizedviral polypeptides of the invention can be inserted recombinantly intothat of a natural or modified (e.g., attenuated) viral genome suitablefor the transduction of a subject (e.g., in vivo administration) orcells isolated from a subject (e.g., for ex vivo transduction followedby administration of the cells back to the subject). Additionalmodifications can be made to the virus to improve infectivity or tropism(e.g., pseudotyping), reduce or eliminate replicative competency, orreduce immunogencity of the viral components (e.g., all components notrelated to the immunogenic vaccine agent). A vector of the invention canbe expressed by the transduced cell and secreted into the extracellularspace or remain with the expressing cell (e.g., as an intracellularmolecule or displayed on the cell surface). Chimeric or pseudotypedviral vectors can also be used to transduce a cell to allow forexpression of one or more optimized viral polypeptides of the invention.Exemplary vectors are described below.

Adenoviruses

Recombinant adenoviruses offer several significant advantages for use asvectors for the expression of one or more optimized viral polypeptidesof the invention. The viruses can be prepared to high titer, can infectnon-replicating cells, and can confer high-efficiency transduction oftarget cells ex vivo following contact with a target cell population.Furthermore, adenoviruses do not integrate their DNA into the hostgenome. Thus, their use as expression vectors has a reduced risk ofinducing spontaneous proliferative disorders. In animal models,adenoviral vectors have generally been found to mediate high-levelexpression for approximately one week. The duration of transgeneexpression (expression of a nucleic acid encoding an optimized viralpolypeptide of the invention) can be prolonged by using cell ortissue-specific promoters. Other improvements in the molecularengineering of the adenoviral vector itself have produced more sustainedtransgene expression and less inflammation. This is seen with so-called“second generation” vectors harboring specific mutations in additionalearly adenoviral genes and “gutless” vectors in which virtually all theviral genes are deleted utilizing a Cre-Lox strategy (Engelhardt et al.,Proc. Natl. Acad. Sci. USA 91:6196 (1994) and Kochanek et al., Proc.Natl. Acad. Sci. USA 93:5731 (1996), each herein incorporated byreference).

The rare serotype and chimeric adenoviral vectors disclosed inInternational Patent Application Publications WO 2006/040330 and WO2007/104792, each incorporated by reference herein, are particularlyuseful as vectors of the invention. For example, recombinantadenoviruses rAd26, rAd34, rAd35, rAd48, and rAd5HVR48 can encode one ormore optimized viral polypeptides of the invention. One or morerecombinant viral vectors encoding optimized viral polypeptides of theinvention can be administered to a subject to treat or prevent a viralinfection.

Adeno-Associated Viruses (AAV)

Adeno-associated viruses (rAAV), derived from non-pathogenicparvoviruses, can also be used to express optimized viral polypeptidesof the invention as these vectors evoke almost no anti-vector cellularimmune response, and produce transgene expression lasting months in mostexperimental systems.

Retroviruses

Retroviruses are useful for the expression of optimized viralpolypeptides of the invention. Unlike adenoviruses, the retroviralgenome is based in RNA. When a retrovirus infects a cell, it willintroduce its RNA together with several enzymes into the cell. The viralRNA molecules from the retrovirus will produce a double-stranded DNAcopy, called a provirus, through a process called reverse transcription.Following transport into the cell nucleus, the proviral DNA isintegrated in a host cell chromosome, permanently altering the genome ofthe transduced cell and any progeny cells that may derive from thiscell. The ability to permanently introduce a gene into a cell ororganism is the defining characteristic of retroviruses used for genetherapy. Retroviruses include lentiviruses, a family of virusesincluding human immunodeficiency virus (HIV) that includes severalaccessory proteins to facilitate viral infection and proviralintegration. Current, “third-generation,” lentiviral vectors featuretotal replication incompetence, broad tropism, and increased genetransfer capacity for mammalian cells (see, e.g., Mangeat and Trono,Human Gene Therapy 16(8):913 (2005) and Wiznerowicz and Trono, TrendsBiotechnol. 23(1):42 (2005), each herein incorporated by reference).

Other Viral Vectors

Besides adenoviral and retroviral vectors, other viral vectors andtechniques are known in the art that can be used to express optimizedviral polypeptides of the invention in a cell (e.g., a blood cell, suchas a lymphocyte) or subject (e.g., a human). Theses viruses includePoxviruses (e.g., vaccinia virus and modified vaccinia virus Ankara or(MVA); see, e.g., U.S. Pat. Nos. 4,603,112 and 5,762,938, eachincorporated by reference herein), Herpesviruses, Togaviruses (e.g.,Venezuelan Equine Encephalitis virus; see, e.g., U.S. Pat. No.5,643,576, incorporated by reference herein), Picornaviruses (e.g.,poliovirus; see, e.g., U.S. Pat. No. 5,639,649, incorporated byreference herein), Baculoviruses, and others described byWattanapitayakul and Bauer (Biomed. Pharmacother. 54:487 (2000),incorporated by reference herein).

Other Expression Vectors: Naked DNA and Oligonucleotides

Naked DNA or oligonucleotides encoding one or more optimized viralpolypeptides of the invention can also be used to express thesepolypeptides in a cell (e.g., a blood cell, such as a lymphocyte) orsubject (e.g., a human). See, e.g., Cohen, Science 259:1691-1692 (1993);Fynan et al., Proc. Natl. Acad. Sci. USA, 90: 11478 (1993); and Wolff etal., BioTechniques 11: 474485 (1991), each herein incorporated byreference. This is the simplest method of non-viral transfection.Efficient methods for delivery of naked DNA exist such aselectroporation and the use of a “gene gun,” which shoots DNA-coatedgold particles into a cell using high pressure gas and carrier particles(e.g., gold).

Lipoplexes and Polyplexes

To improve the delivery of an nucleic acid encoding an optimized viralpolypeptide of the invention into a cell or subject, lipoplexes (e.g.,liposomes) and polyplexes can be used to protect the vector DNA fromundesirable degradation during the transfection process. Plasmid DNA canbe covered with lipids in an organized structure like a micelle or aliposome. When the organized structure is complexed with DNA it iscalled a lipoplex. There are three types of lipids, anionic(negatively-charged), neutral, or cationic (positively-charged).Lipoplexes that utilize cationic lipids have proven utility for genetransfer. Cationic lipids, due to their positive charge, naturallycomplex with the negatively-charged DNA. Also as a result of theircharge they interact with the cell membrane, endocytosis of the lipoplexoccurs, and the DNA is released into the cytoplasm. The cationic lipidsalso protect against degradation of the DNA by the cell.

Complexes of polymers with DNA are called polyplexes. Most polyplexesconsist of cationic polymers and their production is regulated by ionicinteractions. One large difference between the methods of action ofpolyplexes and lipoplexes is that polyplexes cannot release their DNAload into the cytoplasm, so to this end, co-transfection withendosome-lytic agents (to lyse the endosome that is made duringendocytosis) such as inactivated adenovirus must occur. However, this isnot always the case; polymers such as polyethylenimine have their ownmethod of endosome disruption as does chitosan and trimethylchitosan.

Exemplary cationic lipids and polymers that can be used in combinationwith an nucleic acid encoding an optimized viral polypeptide of theinvention to form lipoplexes, or polyplexes include, but are not limitedto, polyethylenimine, lipofectin, lipofectamine, polylysine, chitosan,trimethylchitosan, and alginate.

Hybrid Methods

Several hybrid methods of gene transfer combine two or more techniques.Virosomes, for example, combine lipoplexes (e.g., liposomes) with aninactivated virus. This approach has been shown to result in moreefficient gene transfer in respiratory epithelial cells than eitherviral or liposomal methods alone. Other methods involve mixing otherviral vectors with cationic lipids or hybridising viruses. Each of thesemethods can be used to facilitate transfer of an nucleic acid encodingoptimized viral polypeptides of the invention into a cell (e.g., a bloodcell, such as a lymphocyte) or subject (e.g., a human).

Dendrimers

Dendrimers may be also be used to transfer an nucleic acid encoding anoptimized viral polypeptide of the invention into a cell (e.g., a bloodcell, such as a lymphocyte) or subject (e.g., a human). A dendrimer is ahighly branched macromolecule with a spherical shape. The surface of theparticle may be functionalized in many ways, and many of the propertiesof the resulting construct are determined by its surface. In particularit is possible to construct a cationic dendrimer (i.e. one with apositive surface charge). When in the presence of genetic material suchas DNA or RNA, charge complimentarity leads to a temporary associationof the nucleic acid with the cationic dendrimer. On reaching itsdestination the dendrimer-nucleic acid complex is then taken into thecell via endocytosis.

In Vivo Administration

The invention also features in vivo methods for immunizing a subject(e.g., a human) with a vaccine of the invention. In one embodiment, oneor more vaccines of the invention can be directly administered to asubject to elicit a protective or therapeutic immune response (e.g., acellular or humoral immune response) against a virus (e.g., HIV-1).Alternatively, a vector encoding one or more optimized viralpolypeptides of the invention, as described above, can be directlyadministered to a subject to prevent or treat a viral infection. Avector (e.g., a viral vector) that efficiently transfects or transducesone or more cells in vivo can elicit a broad, durable, and potent immuneresponse in the treated subject. Upon transfer of the nucleic acidcomponent of the expression vector into a host cell (e.g., a blood cell,such as a lymphocyte), the host cell produces and displays or secretesthe vaccine of the invention, which then serves to activate componentsof the immune system such as antigen-presenting cells (APCs), T cells,and B cells, resulting in the establishment of immunity.

Pharmaceutical Compositions

The invention features the vaccines, vectors, and optimized viralpolypeptides of the invention in combination with one or morepharmaceutically acceptable excipients, diluents, buffers, or otheracceptable carriers. The formulation of a vaccine, vector, or optimizedviral polypeptides will employ or allow expression of an effectiveamount of the optimized viral polypeptide immunogen. That is, there willbe included an amount of antigen which will cause the treated subject(e.g., a human) to produce a specific and sufficient immunologicalresponse so as to impart protection to the subject from subsequentexposure to a virus (e.g., HIV-1) or to treat an existing viralinfection. For example, a formulation of a vaccine of the invention canallow for the expression of an amount of antigen which will cause thesubject to produce a broad and specific cellular immune response. Asubject treated with a vaccine, vector, or optimized viral polypeptideof the invention can also produce anti-viral antibodies (e.g.,neutralizing antibodies) which can confer a protective or therapeuticbenefit to the subject. A vaccine, vector, or optimized viralpolypeptide of the invention can be directly administered to a subject,either alone or in combination with any pharmaceutically acceptablecarrier, salt or adjuvant known in the art.

Pharmaceutically acceptable salts may include non-toxic acid additionsalts or metal complexes that are commonly used in the pharmaceuticalindustry. Examples of acid addition salts include organic acids such asacetic, lactic, pamoic, maleic, citric, malic, ascorbic, succinic,benzoic, palmitic, suberic, salicylic, tartaric, methanesulfonic,toluenesulfonic, or trifluoroacetic acids or the like; polymeric acidssuch as tannic acid, carboxymethyl cellulose, or the like; and inorganicacids such as hydrochloric acid, hydrobromic acid, sulfuric acidphosphoric acid, or the like. Metal complexes include zinc, iron, andthe like. One exemplary pharmaceutically acceptable carrier isphysiological saline. Other physiologically acceptable carriers andtheir formulations are known to one skilled in the art and described,for example, in Remington's Pharmaceutical Sciences, (18^(th) edition),ed. A. Gennaro, 1990, Mack Publishing Company, Easton, Pa.

Pharmaceutical formulations of a prophylactically or therapeuticallyeffective amount of a vaccine, vector, or optimized viral polypeptide ofthe invention can be administered orally, parenterally (e.g.,intramuscular, intraperitoneal, intravenous, or subcutaneous injection,inhalation, intradermally, optical drops, or implant), nasally,vaginally, rectally, sublingually, or topically, in admixture with apharmaceutically acceptable carrier adapted for the route ofadministration. The concentration of a vaccine, vector, or optimizedviral polypeptide of the invention in the formulation can vary fromabout 0.1-100 wt. %.

Formulations for parenteral administration of compositions containing avaccine, vector, or optimized viral polypeptide of the invention includesterile aqueous or non-aqueous solutions, suspensions, or emulsions.Examples of suitable vehicles include propylene glycol, polyethyleneglycol, vegetable oils, gelatin, hydrogenated naphalenes, and injectableorganic esters, such as ethyl oleate. Such formulations may also containadjuvants, such as preserving, wetting, emulsifying, and dispersingagents. Biocompatible, biodegradable lactide polymer, lactide/glycolidecopolymer, or polyoxyethylene-polyoxypropylene copolymers may be used tocontrol the release of the compounds. Other potentially usefulparenteral delivery systems for compositions containing a vaccine,vector, or optimized viral polypeptide of the invention includeethylene-vinyl acetate copolymer particles, osmotic pumps, implantableinfusion systems, and liposomes.

Liquid formulations can be sterilized by, for example, filtrationthrough a bacteria-retaining filter, by incorporating sterilizing agentsinto the compositions, or by irradiating or heating the compositions.Alternatively, they can also be manufactured in the form of sterile,solid compositions, which can be dissolved in sterile water or someother sterile injectable medium immediately before use.

Compositions containing vaccine, vector, or optimized viral polypeptideof the invention for rectal or vaginal administration are preferablysuppositories which may contain, in addition to active substances,excipients such as coca butter or a suppository wax. Compositions fornasal or sublingual administration are also prepared with standardexcipients known in the art. Formulations for inhalation may containexcipients, for example, lactose, or may be aqueous solutionscontaining, for example, polyoxyethylene-9-lauryl ether, glycocholateand deoxycholate, or may be oily solutions for administration in theform of nasal drops or spray, or as a gel.

The amount of active ingredient in the compositions of the invention canbe varied. One skilled in the art will appreciate that the exactindividual dosages may be adjusted somewhat depending upon a variety offactors, including the peptide being administered, the time ofadministration, the route of administration, the nature of theformulation, the rate of excretion, the nature of the subject'sconditions, and the age, weight, health, and gender of the patient. Inaddition, the severity of the condition treated by the vaccine, vector,or optimized viral polypeptide will also have an impact on the dosagelevel. Generally, dosage levels of between 0.1 μg/kg to 100 mg/kg ofbody weight are administered daily as a single dose or divided intomultiple doses. Preferably, the general dosage range is between 250μg/kg to 5.0 mg/kg of body weight per day. Wide variations in the neededdosage are to be expected in view of the differing efficiencies of thevarious routes of administration. For instance, oral administrationgenerally would be expected to require higher dosage levels thanadministration by intravenous injection. Variations in these dosagelevels can be adjusted using standard empirical routines foroptimization, which are well known in the art. In general, the preciseprophylactically or therapeutically effective dosage can be determinedby the attending clinician in consideration of the above-identifiedfactors.

The amount of a vaccine, vector, or optimized viral polypeptide of theinvention present in each dose given to a patient is selected withregard to consideration of the patient's age, weight, sex, generalphysical condition and the like. The amount of a vaccine, vector, oroptimized viral polypeptide required to induce an immune response (e.g.,a cellular immune response) or produce an exogenous effect in thepatient without significant adverse side effects varies depending uponthe pharmaceutical composition employed and the optional presence of anadjuvant. Initial doses can be optionally followed by repeated boosts,where desirable. The method can involve chronically administering thevaccine, vector, or optimized viral polypeptide of the invention. Fortherapeutic use or prophylactic use, repeated dosages of the immunizingvaccine, vector, or optimized viral polypeptide can be desirable, suchas a yearly booster or a booster at other intervals. The dosageadministered will, of course, vary depending upon known factors such asthe pharmacodynamic characteristics of the particular vaccine, vector,or optimized viral polypeptide, and its mode and route ofadministration; age, health, and weight of the recipient; nature andextent of symptoms, kind of concurrent treatment, frequency oftreatment, and the effect desired. A vaccine, vector, or optimized viralpolypeptide of the invention can be administered in chronic treatmentsfor subjects at risk of acute infection due to needle sticks or maternalinfection. A dosage frequency for such “acute” infections may range fromdaily dosages to once or twice a week i.v. or i.m., for a duration ofabout 6 weeks. The vaccine, vector, or optimized viral polypeptide canalso be employed in chronic treatments for infected patients, orpatients with advanced infection with a virus (e.g., HIV-1). In infectedpatients, the frequency of chronic administration can range from dailydosages to once or twice a week i.v. or i.m., and may depend upon thehalf-life of immunogen present in the vaccine, vector, or optimizedviral polypeptide of the invention.

Adjuvants

A vaccine of the invention used to vaccinate a mammal (e.g., a human) inneed thereof against a virus can be administered concurrent with or inseries with one or more pharmaceutically acceptable adjuvants toincrease the immunogenicity of the vaccine. Adjuvants approved for humanuse include aluminum salts (alum). These adjuvants have been useful forsome vaccines including hepatitis B, diphtheria, polio, rabies, andinfluenza. Other useful adjuvants include Complete Freund's Adjuvant(CFA), Incomplete Freund's Adjuvant (IFA), muramyl dipeptide (MDP),synthetic analogues of MDP,N-acetylmuramyl-L-alanyl-D-isoglutamyl-L-alanine-2-[1,2-dipalmitoyl-s-gly-cero-3-(hydroxyphosphoryloxy)]ethylamide(MTP-PE) and compositions containing a metabolizable oil and anemulsifying agent, wherein the oil and emulsifying agent are present inthe form of an oil-in-water emulsion having oil droplets substantiallyall of which are less than one micron in diameter.

Kits

The invention provides kits that include a pharmaceutical compositioncontaining a vaccine, vector, or optimized viral polypeptide of theinvention, and a pharmaceutically-acceptable carrier, in atherapeutically effective amount for preventing or treating a viralinfection. The kits include instructions to allow a clinician (e.g., aphysician or nurse) to administer the composition contained therein.

Preferably, the kits include multiple packages of the single-dosepharmaceutical composition(s) containing an effective amount of avaccine, vector, or optimized viral polypeptide of the invention.Optionally, instruments or devices necessary for administering thepharmaceutical composition(s) may be included in the kits. For instance,a kit of this invention may provide one or more pre-filled syringescontaining an effective amount of a vaccine, vector, or optimized viralpolypeptide of the invention. Furthermore, the kits may also includeadditional components such as instructions or administration schedulesfor a patient infected with or at risk of being infected with a virus touse the pharmaceutical composition(s) containing a vaccine, vector, oroptimized viral polypeptide of the invention.

It will be apparent to those skilled in the art that variousmodifications and variations can be made in the compositions, methods,and kits of the present invention without departing from the spirit orscope of the invention. Thus, it is intended that the present inventioncover the modifications and variations of this invention provided theycome within the scope of the appended claims and their equivalents.

EXAMPLES

The present invention is illustrated by the following examples, whichare in no way intended to be limiting of the invention.

Example 1

The mosaic antigen Gag, Pol, Nef, and Env sequences (SEQ ID NOS:1-8)were constructed using the genetic algorithm discussed above. Thesesequences were then modified to make them practical for vaccinedevelopment by eliminating cleavage/fusion activity in Env (SEQ IDNOS:9-11), eliminating catalytic activity in Pol (SEQ ID NOS:12-14),eliminating myristylation sites in Nef (SEQ ID NOS:16-18), andconstructing fusion constructs including GagNef, GagPol, or GagPolNef(SEQ ID NOS:19-29). The comparator optimal natural clade C genes arealso depicted (SEQ ID NOS:30-36).

Example 2

Twenty rhesus monkeys were immunized with 3×10¹⁰ vp rAd26 vectorsexpressing Gag, Pol, and Env genes from M consensus (Group 1), 2-valentM mosaic (Group 2), or optimal natural clade C (Group 3) sequences. TheM consensus sequences represent synthetic sequences that represent thesingle best “average” of circulating viruses worldwide. The 2-valent Mmosaic sequences are described above. The optimal natural clade Csequences are naturally occurring sequences from actual clade C HIV-1viruses that are the most “consensus-like” in character. Cellular immunebreadth was assessed by evaluating the number of responding peptidesfrom the global potential T cell epitope (PTE) peptide set. The PTEpeptides represent >85% of global HIV-1 sequences and are freelyavailable from the NIH.

The results show that the novel 2-valent M mosaic sequences dramaticallyoutperformed these other two leading antigen concepts. As shown in Table1, the 2-valent M mosaic antigens elicited significantly increasedbreadth of Gag-specific, Env-specific, Pol-specific, and total Tlymphocyte responses as compared with M consensus antigens and optimalnatural clade C antigens. (Mean represents the average # epitopes ineach group of monkeys; SEM represents the standard error of the mean).

TABLE 1 Mosaic HIV-1 Gag/Pol/Env Antigens Expand Breadth Against GlobalPTE Peptides in Rhesus Monkeys Group I: M Group II: 2-valent Group III:Natural Consensus M Mosaic Clade C Breadth Mean SEM Mean SEM Mean SEMGag 2.0 0.4 7.7 0.9 2.2 0.5 Env 2.0 0.4 4.0 0.6 1.6 0.5 Pol 2.7 0.5 8.11.4 2.4 0.5 Total 6.7 0.7 19.9 1.9 6.1 1.1

Example 3

Macaque monkeys were immunized IM with 3×10¹⁰ vp rAd26 vectorsexpressing Gag, Pol, and Env genes from M consensus (Group 1; n=7),2-valent M mosaic (Group 2; n=7), or optimal natural clade C (Group 3;n=6) sequences described in Example 2. Cellular immune breadth wasassessed by evaluating the number of responding peptides from the globalpotential T cell epitope (PTE) peptide set.

As a readout, we assessed the CD4/CD8 IFNγ Elispot responses to pooledPTE peptides (magnitude). The epitopes were comprehensively mapped using15 mer PTE peptides to assess the number of positives (positives weredefined as 55 spot forming cells (SFC) per 10⁶ PBMC and 4× background).Pooled sets of overlapping peptides spanning 5 Gag proteins were alsotested to compare responses to a set of complete proteins.

The results show that the 2-valent M mosaic sequences dramaticallyoutperformed the other two leading antigen concepts (Mcon and OptC).

Example 4

We used modeling to validate our observation that T cell responsesincrease as a result of the mosaic vaccine. We fit Poisson regressionmodels that predicted the number of reactive peptides as a function ofvaccine, polypeptide, and T cell type and then did a stepwiseelimination of interactions. We observed that, although the mosaicvaccine produced a highly significant enhancement in the number ofpositive PTE responses, it did so more-or-less uniformly across allpolyproteins and T-cell types. Thus, one may predict the number ofpeptides having a positive effect in an animal by combiningcontributions that depend, separately, on the type of T-cell, thepolypeptide, and the vaccine the animal received.

These models also included random effects to account foranimal-to-animal variation. This is a precaution designed to make formore credible p-values, by properly apportioning the predictive power ofthe model.

We observed the following effects:

a) There are many more CD8 responses than CD4 responses, by a factor of4.37, p<2×10⁻¹⁶;

b) There are fewer responses in gp 160 than in gag or pol, by a factorof 0.54, p=0.000830, and no significant difference between gag and pol(even when normalized by sequence length as pol is twice as long as Gagand so has more opportunity to react); and

c) The mosaic vaccine generates significantly more positive responsesthan Mcon (by a factor of 3.6, p=6.26×10⁻¹¹) while OptC generates fewer,though the Mcon-OptC difference is not significant.

Example 5

If one considers just the minimal number of responses elicited by avaccine and detected by PTE peptides, so that all peptide that overlapby >8 amino acids regardless of variation are counted just 1 time, themosaic vaccines still generate a greater number of responses to distinctregions.

For CD8, counting each overlapping peptide set just once:

Statistical Summary:

Mos2>Mcon˜OptC (Mcon shows a trend for more response than OptC)

Wilcoxon p-value for Mos2 compared to Mcon: p-value=0.0009992

Wilcoxon p-value for Mcon compared to Optimal C: p-value=0.2351

Summary of the Groups:

Vaccine Min. 1stQu. Median Mean 3rdQu. Max. Mos2.cd8 7 7.5 8 9.4 11 14Mcon.cd8 0 3 3 3.3 4 6 OptC.cd8 0 1 1.5 2 4.25 5For CD4, counting each overlapping peptide set just once (there is verylittle overlap in CD4, so this is almost the same as the first count).Statistical Summary:Mos2>>Mcon˜OptC (Mcon shows a trend for more response than OptC)Wilcoxon p-value for Mos2 compared to Mcon: p-value=0.00198Wilcoxon p-value for Mcon compared to Optimal C, 0.099Summary of the Groups:

Vaccine Min. 1stQu. Median Mean 3rdQu. Max. Mos2.cd4 2 2.5 3 3.4 4.5 5Mcon.cd4 0 1 1 1.3 2 2 OptC.cd4 0 0 0.5 0.67 1 2

Example 6 Poisson Regression Counting Each Overlapping Peptide Set JustOnce

Using overlapping PTE peptides, we determined the following, which arein broad agreement with the results discussed in Example 4 above, whereeach positive PTE response counted separately:

a) There are many more CD8 responses than CD4 responses, by a factor ofabout 2.8, p≈1×10⁻⁷;

b) The mosaic vaccine generates significantly more positive responsesthan Mcon (by a factor of 2.84, p≈4.3×10⁻⁷), while OptC generates fewer,though the Mcon-OptC difference is not significant; and

c) There are more responses to Pol than to Gag and more to Gag thangp160, but only the Pol-gp160 difference, a factor of about 2, wassignificant, p<0.001.

Example 7

The following table is a tally of the total responses to Gag, Pol, andEnv responses to the three vaccines in the 7 animals vaccinated with 2Mosaic (Mos2) or Mcon, and the 6 animals vaccinated with the OptimalNatural C clade (OptC):

CD8 CD4 Env Gag Pol Env Gag Pol 2Mos 13 20 33 3 10 11 ConM 8 7 8 2 3 4OptC 4 5 5 1 2 1The OptC vaccine yielded an average response across all monkeys that wasslightly less than the CD8+ T cell response per protein. The Mconvaccine exhibited ˜1 response per protein. Only with Mos2 do we observea difference in the proteins, where Env typically has fewer responsesthan either Gag or Pol.

Each of the proteins in the Mos2 vaccine elicited many responses andcontributed to the overall response. The relative length of theconsensus proteins after the modifications to inactivate pol and thedeletion of the cleavage and fusion domain in Env was: 671 amino acidsof Env, 851 of Pol, 498 of Gag (1.35:1.7:1).

Summary

Breadth: The 2 mosaic vaccines elicit T cell responses that are capableof recognizing many more epitope-regions than the M consensus or asingle optimal natural strain.

Depth: The diversity of the PTE peptides recognized suggests both formsin the 2 mosaics are eliciting different T cell responses to the variantpeptides, increasing the cross-reactive potential.

Example 8

Mosaic HIV-1 vaccines of the invention expand the breadth and depth ofcellular immune responses in Rhesus monkeys. We constructed mosaic HIV-1Gag, Pol, and Env antigens that optimized PTE coverage of HIV-1 M groupsequences, which include all major HIV-1 clades and recombinant lineagesin the Los Alamos HIV-1 sequence

database. A 2-valent mosaic strategy was utilized to balance thecompeting issues of theoretical coverage and practical utility. 2-valentmosaic HIV-1 Gag, Pol, and Env antigens substantially expanded thebreadth and magnitude (depth) of epitope-specific CD8+ and CD4+ Tlymphocyte responses in rhesus monkeys, relative to the immune responseobserved using consensus and natural sequence HIV-1 antigens in rhesusmonkeys.

We immunized 27 outbred rhesus monkeys with a single injection ofrecombinant adenovirus serotype 26 (rAd26) vectors expressing thefollowing antigens: (i) 2-valent mosaic (N=7), (ii) M consensus (N=7),(iii) 2-valent combined clade B and clade C(N=7), or (iv) optimalnatural clade C(N=6) HIV-1 Gag, Pol, and Env antigens. A total dose of3×10¹⁰ viral particles of rAd26 vectors expressing these antigens wasadministered once i.m. to each animal. The optimal clade C antigens werethe natural strain sequences selected to provide maximal PTE coverage ofclade C sequences in the Los Alamos HIV-1 sequence database (discussedin the Materials and Methods below). We assessed the breadth andmagnitude (depth) of vaccine-elicited HIV-1-specific T lymphocyteresponses by IFN-γ ELISPOT assays at week 4 following immunizationutilizing pools and subpools of peptides that included all PTEs found inat least 15% of HIV-1 M group sequences. All individual peptideresponses were resolved, and cell-depleted IFN-γ ELISPOT assays wereperformed to determine if reactive peptides represented CD8+ or CD4+ Tlymphocyte epitopes.

The total number of Gag-, Pol-, and Env-specific cellular immuneresponses to PTE peptides elicited by the mosaic antigens was 3.8-foldhigher than the number of responses induced by the consensus or naturalsequence antigens (FIG. 19A; P=1×10-11, comparing the mosaic with theconsensus antigens, the next highest group, based on a Poissonregression model). There were 4.4-fold more CD8+ than CD4+ T lymphocyteresponses (P<10-11) and fewer responses to Env than to Gag or Pol(P<0.0007). The median number of CD8+ T lymphocyte responses was highestfor the mosaic vaccine, followed by the consensus, the combined B+C, andthe natural clade C vaccines (medians of 16, 5, 3, and 2 responses peranimal in each group, respectively). Although there were fewer CD4+ Tlymphocyte responses overall, the same relative pattern emerged with thehighest number of CD4+ T lymphocyte responses to the mosaic vaccine,followed by the consensus, the combined B+C, and the natural clade Cvaccines (medians of 4, 1, 1, and 0.5 responses per animal in eachgroup, respectively). The numbers of CD8+ and CD4+ T lymphocyteresponses elicited by the consensus, the combined B+C, and the naturalclade C vaccines were not statistically distinguishable.

PTE peptides include multiple overlapping sequences that reflectnaturally occurring HIV-1 sequence polymorphisms, and thus the PTEpeptide responses encompass both the recognition of a particular epitope(breadth) and the cross-recognition of variants of that epitope (depth).We performed a conservative analysis of breadth by assessing the numberof reactive epitopic regions per monkey in which all reactive PTEpeptides that overlapped by 8 or more amino acids were counted as oneevent. In this conservative analysis, we still observed that the mosaicantigens elicited 3.1-fold increased numbers of Gag, Pol, and Envreactive epitopic regions as compared with the consensus antigens ornatural sequence antigens (FIG. 19B; P=1.6×10-7, Poisson regression).Epitopic regions exhibited some clustering across animals, as evidencedby regions of high epitope density (FIGS. 20A-20C and FIGS. 21A-21C).Complete alignments of all positive peptides organized by responseregions are shown in FIG. 22.

These data show that the mosaic antigens substantially increased thebreadth of cellular immune responses as compared with M consensus andnatural clade C antigens. The 2-valent mosaic antigens also provedsuperior to the 2-valent combination of clade B and clade C antigens(FIGS. 19A and 19B), indicating that the enhanced breadth was due to themosaic sequence design and did not simply reflect the use of twodistinct antigenic sequences per protein. To determine if the increasedbreadth induced by mosaic antigens compromised the potency of theresponses, we assessed the magnitude of all individual CD8+ and CD4+ Tlymphocyte responses. The magnitude of these responses proved comparableamong all groups (FIG. 23; P=0.58 and P=0.99, respectively, two-sidedKolmogorov-Smirnov tests). Thus, mosaic antigens expanded cellularimmune breadth without compromising the magnitude of individualepitope-specific responses, indicating that antigenic competition andimmunodominance constraints did not limit the immunogenicity of themosaic antigens in this study.

We next characterized the depth of the cellular immune responseselicited by the various vaccine regimens. We defined depth as the numberof simultaneously elicited variant PTE peptides for a particularepitopic region. Inducing responses to multiple common epitope variantsmay increase immunologic coverage of infecting virus sequences, blockcommon escape routes in vivo, or force the virus down tertiary escaperoutes that incur high fitness costs. The consensus and natural sequenceantigens elicited responses that were characterized by a high degree ofsequence identity between the vaccine sequences and the reactive PTEpeptides, as exemplified by the responses in monkey 366 that receivedthe natural clade C antigens (FIG. 24A; see also FIG. 22). In contrast,the mosaic antigens elicited responses that were characterized bymultiple reactive PTE peptides in particular epitopic regions. Thesepeptides represented common variants and often reflected thepolymorphisms contained in the mosaic vaccine sequences, as exemplifiedby the responses in monkey 361 (FIG. 24B; see also FIG. 22). A summaryof all epitope-specific responses in these animals demonstrates that themosaic antigens increased the frequency of cellular immune responses topeptides with two or more targeted variants as compared with theconsensus or natural sequence antigens (FIG. 24C; P=0.001, Wilcoxonrank-sum test comparing the mosaic with the consensus antigens, the nexthighest group).

To complement the analysis utilizing PTE peptides, we also assessed thebreadth of cellular immune responses in the vaccinated monkeys withtraditional overlapping peptides covering 5 different Gag sequences:clade C DU422, clade C ZM651, consensus C, consensus A, and consensus B.Cellular immune breadth was determined by assessing reactivity tosubpools of 10 overlapping peptides spanning each Gag sequence. Themosaic antigens elicited greater breadth of T lymphocyte responses ascompared with the consensus or natural sequence antigens against all Gagsequences that were tested (FIG. 25; P=1×10⁻⁷, binomial regression).Thus, the mosaic antigens augmented cellular immune breadth not only toPTE peptides but also to actual Gag peptides from clades A, B, and C.The mosaic antigens even proved superior to the optimal natural clade Cantigens for inducing responses against clade C Gag peptides. Moreover,the mosaic antigens elicited comparable responses to Gag peptides frommultiple clades, whereas the natural clade C antigens exhibiteddiminished responses to clade A and clade B Gag peptides (FIG. 25).

To assess the durability of these observations, we boosted the monkeysthat received the mosaic, consensus, and optimal natural clade Cantigens at week 40 with a total dose of 3×10¹⁰ viral particles of theheterologous vector rAd5HVR48 expressing HIV-1 Gag, Pol, and Envantigens that matched the sequences utilized in the initialimmunization. Cellular immune breadth was determined by assessingreactivity to subpools of 10 PTE peptides at week 4 (post-prime) and atweek 44 (post-boost). The majority of CD8+ and CD4+ T lymphocyteresponses that were observed after the priming immunization expandedfollowing the boost (FIG. 26A, red and blue lines), and a number of newresponses were also detected (FIG. 26A, red and blue dots). At week 44,the magnitude of individual cellular immune responses proved comparableamong groups (FIG. 26A). The number of subpool responses elicited by themosaic antigens (median 27 responses per animal), however, remainedsubstantially higher than the number of subpool responses induced by theconsensus antigens (median 11 responses per animal) or the optimalnatural clade C antigens (median 10 responses per animal) following theboost immunization (FIG. 26B). Both before and after the boost, therewere more responses per animal elicited by the mosaic vaccine than bythe consensus or natural clade C vaccines (P<0.001, Wilcoxon rank-sumtests for all pairwise comparisons).

We also measured Env-specific humoral immune responses following theboost immunization by ELISAs (FIG. 26C) and luciferase-based pseudovirusneutralization assays (FIG. 26D). All groups exhibited comparable ELISAtiters to clade C gp140 and comparable neutralizing antibody (NAb)responses to the tier 1 clade C virus MW965.26. The mosaic antigenselicited slightly higher Nab responses to the tier 1 clade B virusSF162.LS as compared with the consensus or natural clade C antigens(P=0.02, Wilcoxon rank-sum test), although we did not detect any NAbresponses to tier 2 viruses in any group.

Our data demonstrate that mosaic HIV-1 Gag, Pol, and Env antigensaugmented both the breadth and depth of epitope-specific cellular immuneresponses as compared with consensus or natural sequence antigens inrhesus monkeys, in good agreement with theoretical predictions (FIG.27). The striking results with mosaic antigens in this study may havereflected the fact that rAd26 vectors are particularly efficient ateliciting CD8+ T lymphocyte responses as well as the fact that mosaicantigens appear particularly effective at augmenting CD8+ T lymphocytebreadth (FIGS. 19A and 19B). We also observed enhanced CD4+ T lymphocytebreadth with mosaic antigens, although there were substantially lowernumbers of these responses.

The breadth of Gag-specific cellular immune responses has been shown tobe critical for SIV control in rhesus monkeys and for HIV-1 control inhumans. Moreover, in the phase 2b STEP study, the rAd5-based HIV-1vaccine candidate expressing natural clade B Gag, Pol, and Nef antigenselicited only a limited breadth of HIV-1-specific cellular immuneresponses, and no vaccine benefit was observed. Vaccinees in the STEPstudy developed a median of only 2-3 epitope-specific T lymphocyteresponses, including a median of only 1 epitope-specific response toGag, and this very narrow breadth of cellular immune responses likelyprovided insufficient immunologic coverage of the diversity of infectingviruses. Viral escape from CD8+ T lymphocytes has also been reported tooccur rapidly during acute HIV-1 infection, and thus vaccine-elicitedcellular immune responses against common epitope variants may also provecritical. Taken together, these studies emphasize the need to developHIV-1 vaccine strategies that augment cellular immune breadth and depth.

Since we evaluated mosaic HIV-1 antigens in the present study, we wereunable to assess the protective efficacy of these vaccine regimensagainst SIV challenges. However, we have previously reported that thebreadth of SIV-specific cellular immune responses elicited by rAdvectors correlated with protective efficacy against SIV challenges inrhesus monkeys (Liu et al., Nature 457:87, 2009). We have also shownthat cellular immune responses against variant epitopes can block SIVmutational evolution in rhesus monkeys in vivo (Barouch et al., Nat.Immunol. 6:247, 2005), suggesting the biologic relevance of expandingcellular immune depth. Modeling the protective efficacy of mosaicvaccines against SIV challenges in nonhuman primates has intrinsiclimitations, since the observed diversity of SIV and HIV-1 M groupsequences differs substantially and is influenced by differentunderlying biology. For example, CD8+ T lymphocyte selection pressure innatural hosts such as sooty mangabees appears substantially less thanthat in humans. Thus, the further evaluation of mosaic antigens ascandidate HIV-1 vaccines can be benefited by clinical trials.

In summary, we demonstrate that 2-valent mosaic HIV-1 Gag, Pol, and Envantigens substantially expanded cellular immune breadth and depth inrhesus monkeys. These findings have major implications for HIV-1 vaccinedevelopment, since global virus diversity and viral escape from cellularimmune responses represent critical hurdles in the development of a Tcell-based HIV-1 vaccine. A 2-valent cocktail of mosaic antigens is alsopractical and potentially feasible for clinical development. Increasingthe valency of mosaic antigens may further improve coverage. Finally,the mosaic antigen strategy is generalizable and could be utilized forother genetically diverse pathogens in addition to HIV-1.

Materials and Methods

Antigen design and vector production. 2-valent mosaic Gag, Pol, and Envantigens were constructed to provide optimal coverage of HIV-1 M groupsequences in the Los Alamos HIV-1 sequence database essentially asdescribed (1, 2). Optimal natural clade C antigens were selected to bethe sequences that provide optimal PTE coverage of clade C sequences inthe Los Alamos HIV-1 sequence database (C.IN.-0.70177 Gag,C.ZA.04.04ZASK208B1 Pol, C.SN.90.90SE 364 Env). Clade B antigens wereselected to be near-consensus or consensus sequences (B.CAM-1 Gag,B.IIIB Pol, B.Con Env) and were used to complement the optimal clade Cantigens for the 2-valent clade B+C vaccine approach. Pol antigenscontained RT and IN without PR and included point mutations to eliminatecatalytic activity as described (Priddy et al., Clinical infectiousdiseases 46:1769, 2008). Env gp140 antigens contained point mutations toeliminate cleavage and fusion activity. Vaccine sequences are depictedin FIG. 27. Recombinant, replication-incompetent adenovirus serotype 26(rAd26) and hexon-chimeric rAd5HVR48 vectors expressing these antigenswere grown in PER.55K cells and purified by double CsC1 gradientsedimentation essentially as described (Abbink et al., J. Virol.81:4654, 2007, and Roberts et al., Nature 441:239, 2006).

Animals and immunizations. 27 outbred rhesus monkeys that did notexpress the MHC class I allele Mamu-A*01 were housed at New EnglandPrimate Research Center (NEPRC), Southborough, Mass. Immunizationsinvolved 3×10¹⁰ viral particles rAd26 or rAd5HVR48 vectors expressingmosaic, M consensus, clade B+clade C, or optimal natural clade C HIV-1Gag, Pol, and Env antigens delivered as 1 ml injections i.m. in bothquadriceps muscles at weeks 0 and 40. All animal studies were approvedby our Institutional Animal Care and Use Committees (IACUC).

IFN-γ ELISPOT assays. HIV-1-specific cellular immune responses invaccinated monkeys were assessed by interferon-γ (IFN-γ) ELISPOT assaysessentially as described (Roberts et al., Nature 441:239, 2006, and Liuet al., Nature 457:87, 2009). HIV-1 Gag, Pol, and Env potential T cellepitope (PTE) peptides that included all PTEs found in at least 15% ofHIV-1 M group sequences as well as HIV-1 Gag peptides from clade CDU422, clade C ZM651, consensus C, consensus A, and consensus B strainswere obtained from the NIH AIDS Research and Reference Reagent Program.96-well multiscreen plates (Millipore) were coated overnight with 100 of10 μg/ml anti-human IFN-γ (BD Biosciences) in endotoxin-free Dulbecco'sPBS (D-PBS). The plates were then washed three times with D-PBScontaining 0.25% Tween-20 (D-PBS/Tween), blocked for 2 h with D-PBScontaining 5% FBS at 37° C., washed three times with D-PBS/Tween, rinsedwith RPMI 1640 containing 10% FBS to remove the Tween-20, and incubatedwith 2 μg/ml each peptide and 2×10⁵ PBMC in triplicate in 100 μlreaction volumes. Following an 18 h incubation at 37° C., the plateswere washed nine times with PBS/Tween and once with distilled water. Theplates were then incubated with 2 μg/ml biotinylated anti-human IFN-γ(BD Biosciences) for 2 h at room temperature, washed six times withPBS/Tween, and incubated for 2 h with a 1:500 dilution ofstreptavidin-alkaline phosphatase (Southern Biotechnology Associates).Following five washes with PBS/Tween and one with PBS, the plates weredeveloped with nitro bluetetrazolium/5-bromo-4-chloro-3-indolyl-phosphate chromogen (Pierce),stopped by washing with tap water, air dried, and read using an ELISPOTreader (Cellular Technology Ltd). Spot-forming cells (SFC) per 10⁶ PBMCwere calculated. Media backgrounds were typically <15 SFC per 10⁶ PBMC.Positive responses were defined as >55 SFC per 10⁶ PBMC and >4-foldbackground.

Epitope mapping. Comprehensive CD8+ and CD4+ T lymphocyte epitopemapping was performed utilizing Gag, Pol, and Env PTE peptides that wereobtained from the NIH AIDS Research and Reference Reagent Program. IFN-γELISPOT assays were conducted at week 4 following immunization initiallywith complete peptide pools as well as with subpools containing 10 PTEpeptides. All peptide subpools with positive responses weredeconvoluted, and epitopes were confirmed with individual 15 amino acidPTE peptides. Cell-depleted IFN-γ ELISPOT assays were then performed todetermine if reactive peptides represented CD8+ or CD4+ T lymphocyteepitopes. Partial epitope mapping utilizing PTE subpools was alsoperformed 4 weeks following the boost immunization at week 44. Allborderline responses were retested and only considered positive ifconfirmed. Partial epitope mapping utilizing subpools containing 10overlapping Gag peptides was also performed to assess breadth to HIV-1Gag from various clades.

Humoral immune assays. Env-specific humoral immune responses wereevaluated by direct ELISAs utilizing HIV-1 clade C Env gp140 andluciferase-based pseudovirus neutralization assays essentially asdescribed (Montefiori, Evaluating neutralizing antibodies against HIV,SIV and SHIV in luciferase reporter gene assays. Current Protocols inImmunology, Coligan, Kruisbeek, Margulies, Shevach, Strober, and Coico,Ed. (John Wiley & Sons, 2004, pp. 1-15).

Statistical analyses. All statistical analyses were done using thepackage R (Team, Foundation for Statisical Computing, Vienna, Austria,2009). To analyze the breadth of cellular immune responses to mapped PTEpeptides (FIG. 19A), we fit Poisson regression models that predicted thenumber of reactive peptides as a function of vaccine group, antigen(Gag, Pol, Env), and lymphocyte subpopulation (CD4, CD8). Our modelsincluded random effects to accommodate animal-to-animal variation andwere fit with the lme4 library (Pinheiro, Springer, N.Y. (2000)) of thepackage R. The data fit the models well (dispersion parameter 1.0), andthere were no significant interactions among the three explanatoryfactors. For example, the 3.8-fold enhancement in the number of PTEpeptides recognized by monkeys that received the mosaic antigens ascompared to those that received the consensus or natural sequenceantigens (FIG. 19A) applied equally to PTEs from Gag, Pol, and Env andheld for responses by CD8+ as well as CD4+ T lymphocytes. The analysisof the number of reactive epitopic regions (FIG. 19B) also includedPoisson regression models with random effects and again fit well(dispersion parameter 0.87) without any significant interactions.Comparisons of the magnitude of CD8+ and CD4+ T lymphocyte responses(FIG. 23) were performed utilizing 2-sided Kolmogorov-Smirnov tests.Non-parametric tests to compare the breadth and depth of responses permonkey between different vaccines were also performed (FIGS. 19A and24C). We initially employed Kruskal-Wallis tests to determine if therewas a difference among the 4 vaccine groups. In each case this washighly significant, and we then assessed all pairwise comparisonsbetween the 4 vaccine groups using Wilcoxon rank-sum tests. In each ofthese comparisons, the mosaic vaccine elicited significantly moreresponses per monkey than the other 3 vaccines. To analyze the breadthof responses to HIV-1 Gag from various clades (FIG. 25), we fit the datato binomial regression models. These models used the vaccine group as anexplanatory variable and included random effects to account foranimal-to-animal and strain-to-strain variation. The data were slightlyunderdispersed, but the animals that received the mosaic vaccine stillelicited a significantly larger number of responses. PTE coverageassessment was performed using tools available at the Los Alamos HIV-1sequence database.

SEQUENCE APPENDIX I. 2-VALENT M MOSAIC ENV GP160, GAG, POL, NEFSEQUENCES MOSAIC ENV1 GP160 (AA SEQUENCE) SEQ ID NO: 1MRVTGIRKNYQHLWRWGTMLLGILMICSAAGKLWVTVYYGVPVWKEATTTLFCASDAKAYDTEVHNVWATHACVPTDPNPQEVVLENVTENFNMWKNNMVEQMHEDIISLWDQSLKPCVKLTPLCVTLNCTDDVRNVTNNATNTNSSWGEPMEKGEIKNCSFNITTSIRNKVQKQYALFYKLDVVPIDNDSNNTNYRLISCNTSVITQACPKVSFEPIPIHYCAPAGFAILKCNDKKFNGTGPCTNVSTVQCTHGIRPVVSTQLLLNGSLAEEEVVIRSENFTNNAKTIMVQLNVSVEINCTRPNNNTRKSIHIGPGRAFYTAGDIIGDIRQAHCNISRANWNNTLRQIVEKLGKQFGNNKTIVFNHSSGGDPEIVMHSFNCGGEFFYCNSTKLFNSTWTWNNSTWNNTKRSNDTEEHITLPCRIKQIINMWQEVGKAMYAPPIRGQIRCSSNITGLLLTRDGGNDTSGTEIFRPGGGDMRDNWRSELYKYKVVKIEPLGVAPTKAKRRVVQREKRAVGIGAVFLGFLGAAGSTMGAASMTLTVQARLLLSGIVQQQNNLLRAIEAQQHLLQLTVWGIKQLQARVLAVERYLKDQQLLGIWGCSGKLICTTTVPWNASWSNKSLDKIWNNMTWMEWEREINNYTSLIYTLIEESQNQQEKNEQELLELDKWASLWNWFDISNWLWYIKIFIMIVGGLVGLRIVFAVLSIVNRVRQGYSPLSFQTRLPAPRGPDRPEGIEEEGGERDRDRSVRLVDGFLVLIWDDLQSLCLFSYHRLRDLLLIVELLGRRGWEALKYWWNLLQYWSQELKNSAISLLNATAVAVAEGTDRVIEALQRACRAILHIPRRIRQ GLERLLLMOSAIC ENV2 GP160 (AA SEQUENCE) SEQ ID NO: 2MRVRGIQRNWPQWWIWGILGFWMIIICRVMGNLWVTVYYGVPVWKEAKTTLFCASDAKAYEKEVHNVWATHACVPTDPNPQEMVLENVTENFNMWKNDMVDQMHEDIIRLWDQSLKPCVKLTPLCVTLECRNVRNVSSNGTYNIIHNETYKEMKNCSFNATTVVEDRKQKVHALFYRLDIVPLDENNSSEKSSENSSEYYRLINCNTSAITQACPKVSFDPIPIHYCAPAGYAILKCNNKTFNGTGPCNNVSTVQCTHGIKPVVSTQLLLNGSLAEEEIIIRSENLTNNAKTIIVHLNETVNITCTRPNNNTRKSIRIGPGQTFYATGDIIGDIRQAHCNLSRDGWNKTLQGVKKKLAEHFPNKTINFTSSSGGDLEITTHSFNCRGEFFYCNTSGLFNGTYMPNGTNSNSSSNITLPCRIKQIINMWQEVGRAMYAPPIAGNITCRSNITGLLLTRDGGSNNGVPNDTETFRPGGGDMRNNWRSELYKYKVVEVKPLGVAPTEAKRRVVEREKRAVGIGAVFLGILGAAGSTMGAASITLTVQARQLLSGIVQQQSNLLRAIEAQQHMLQLTVWGIKQLQTRVLAIERYLQDQQLLGLWGCSGKLICTTAVPWNTSWSNKSQTDIWDNMTWMQWDKEIGNYTGEIYRLLEESQNQQEKNEKDLLALDSWKNLWNWFDITNWLWYIKIFIMIVGGLIGLRIILGVLSIVRRVRQGYSPLSFQTLTPNPRGLDRLGRIEEEGGEQDRDRSIRLVNGFLALAWDDLRSLCLFSYHQLRDFILIVARAVELLGRSSLRGLQRGWEALKYLGNLVQYWGLELKKGAISLLDTIAIAVAEGTDRIIELIQSICRA IRNIPRRIRQGFEASLLMOSAIC GAG1 (AA SEQUENCE) SEQ ID NO: 3MGARASVLSGGELDRWEKIRLRPGGKKKYRLKHIVWASRELERFAVNPGLLETSEGCRQILGQLQPSLQTGSEELRSLYNTVATLYCVHQRIEIKDTKEALEKIEEEQNKSKKKAQQAAADTGNSSQVSQNYPIVQNIQGQMVHQAISPRTLNAWVKVVEEKAFSPEVIPMFSALSEGATPQDLNTMLNTVGGHQAAMQMLKETINEEAAEWDRVHPVHAGPIAPGQMREPRGSDIAGTTSTLQEQIGWMTNNPPIPVGEIYKRWIILGLNKIVRMYSPVSILDIRQGPKEPFRDYVDRFYKTLRAEQASQDVKNWMTETLLVQNANPDCKTILKALGPAATLEEMMTACQGVGGPGHKARVLAEAMSQVTNSATIMMQRGNFRNQRKTVKCFNCGKEGHIAKNCRAPRKKGCWKCGKEGHQMKDCTERQANFLGKIWPSNKGRPGNFLQNRPEPTAPPEESFRFGEETTTPSQKQEPIDKEMYPLASLKSLFGNDPSSQMOSAIC GAG2 (AA SEQUENCE) SEQ ID NO: 4MGARASILRGGKLDKWEKIRLRPGGKKHYMLKHLVWASRELERFALNPGLLETSEGCKQIIKQLQPALQTGTEELRSLFNTVATLYCVHAEIEVRDTKEALDKIEEEQNKSQQKTQQAKEADGKVSQNYPIVQNLQGQMVHQPISPRTLNAWVKVIEEKAFSPEVIPMFTALSEGATPQDLNTMLNTVGGHQAAMQMLKDTINEEAAEWDRLHPVHAGPVAPGQMREPRGSDIAGTTSNLQEQIAWMTSNPPIPVGDIYKRWIILGLNKIVRMYSPTSILDIKQGPKEPFRDYVDRFFKTLRAEQATQDVKNWMTDTLLVQNANPDCKTILRALGPGATLEEMMTACQGVGGPSHKARVLAEAMSQTNSTILMQRSNFKGSKRIVKCFNCGKEGHIARNCRAPRKKGCWKCGKEGHQMKDCTERQANFLGKIWPSHKGRPGNFLQSRPEPTAPPAESFRFEETTPAPKQEPKDREPLTSLRSLFGSDPLSQ MOSAIC POL1 (AA SEQUENCE)SEQ ID NO: 5 FFRENLAFQQGEAREFPSEQTRANSPTSRELQVRGDNPHSEAGAERQGTLNFPQITLWQRPLVSIKVGGQIREALLDTGADDTVLEDINLPGKWKPKMIGGIGGFIKVRQYDQILIEICGKKAIGTVLVGPTPVNIIGRNMLTQLGCTLNFPISPIETVPVKLKPGMDGPRVKQWPLTEEKIKALTAICEEMEKEGKITKIGPENPYNTPVFAIKKKDSTKWRKLVDFRELNKRTQDFWEVQLGIPHPAGLKKKKSVTVLDVGDAYFSVPLDEGFRKYTAFTIPSTNNETPGIRYQYNVLPQGWKGSPAIFQCSMTRILEPFRAKNPEIVIYQYMDDLYVGSDLEIGQHRAKIEELREHLLKWGFTTPDKKHQKEPPFLWMGYELHPDKWTVQPIQLPEKDSWTVNDIQKLVGKLNWASQIYPGIKVRQLCKLLRGAKALTDIVPLTEEAELELAENREILKEPVHGVYYDPSKDLIAEIQKQGHDQWTYQIYQEPFKNLKTGKYAKMRTAHTNDVKQLTEAVQKIAMESIVIWGKTPKFRLPIQKETWETWWTDYWQATWIPEWEFVNTPPLVKLWYQLEKDPIAGVETFYVDGAANRETKLGKAGYVTDRGRQKIVSLTETTNQKTELQAIYLALQDSGSEVNIVTDSQYALGIIQAQPDKSESELVNQIIEQLIKKERVYLSWVPAHKGIGGNEQVDKLVSSGIRKVLFLDGIDKAQEEHEKYHSNWRAMASDFNLPPVVAKEIVASCDQCQLKGEAMHGQVDCSPGIWQLDCTHLEGKIILVAVHVASGYIEAEVIPAETGQETAYFILKLAGRWPVKVIHTDNGSNFTSAAVKAACWWAGIQQEFGIPYNPQSQGVVESMNKELKKIIGQVRDQAEHLKTAVQMAVFIHNFKRKGGIGGYSAGERIIDIIATDIQTKELQKQIIKIQNFRVYYRDSRDPIWKGPAKLLWKGEGAVVIQDNSDIKVVPRRKVKIIKDYGKQMAGADCVAGRQDEDMOSAIC POL2 (AA SEQUENCE) SEQ ID NO: 6FFRENLAFPQGKAREFSSEQTRANSPTRRELQVWGRDNNSLSEAGADRQGTVSFSFPQITLWQRPLVTIKIGGQLKEALLDTGADDTVLEEMNLPGRWKPKMIGGIGGFIKVRQYDQIPIEICGHKAIGTVLVGPTPVNIIGRNLLTQIGCTLNFPISPIETVPVKLKPGMDGPKVKQWPLTEEKIKALVEICTEMEKEGKISKIGPENPYNTPIFAIKKKDSTKWRKLVDFRELNKRTQDFWEVQLGIPHPAGLKKKKSVTVLDVGDAYFSVPLDEDFRKYTAFTIPSINNETPGIRYQYNVLPQGWKGSPAIFQSSMTKILEPFRKQNPDIVIYQYMDDLYVGSDLEIGQHRTKIEELRQHLLRWGFTTPDKKHQKEPPFLWMGYELHPDKWTVQPIVLPEKDSWTVNDIQKLVGKLNWASQIYAGIKVKQLCKLLRGTKALTEVVPLTEEAELELAENREILKEPVHGVYYDPSKDLIAEIQKQGQGQWTYQIYQEPFKNLKTGKYARMRGAHTNDVKQLTEAVQKIATESIVIWGKTPKFKLPIQKETWEAWWTEYWQATWIPEWEFVNTPPLVKLWYQLEKEPIVGAETFYVDGAANRETKLGKAGYVTDRGRQKVVSLTDTTNQKTELQAIHLALQDSGLEVNIVTDSQYALGIIQAQPDKSESELVSQIIEQLIKKEKVYLAWVPAHKGIGGNEQVDKLVSRGIRKVLFLDGIDKAQEEHEKYHSNWRAMASEFNLPPIVAKEIVASCDKCQLKGEAIHGQVDCSPGIWQLDCTHLEGKVILVAVHVASGYIEAEVIPAETGQETAYFLLKLAGRWPVKTIHTDNGSNFTSATVKAACWWAGIKQEFGIPYNPQSQGVVESINKELKKIIGQVRDQAEHLKTAVQMAVFIHNFKRKGGIGEYSAGERIVDIIASDIQTKELQKQITKIQNFRVYYRDSRDPLWKGPAKLLWKGEGAVVIQDNSDIKVVPRRKAKIIRDYGKQMAGDDCVASRQ DEDMOSAIC NEF1 (AA SEQUENCE) SEQ ID NO: 7MGGKWSKSSVVGWPAIRERMRRAEPAADGVGAVSRDLEKHGAITSSNTAANNADCAWLEAQEEEEVGFPVRPQVPLRPMTYKGALDLSHFLKEKGGLEGLIYSQKRQDILDLWVYHTQGYFPDWQNYTPGPGIRYPLTFGWCFKLVPVEPEKIEEANEGENNSLLHPMSQHGMDDPEKEVLMWKFDSRLAFHHMARELHP EYYKDCMOSAIC NEF2 (AA SEQUENCE) SEQ ID NO: 8MGGKWSKSSIVGWPAVRERIRRAEPAAEGVGAASQDLDKYGALTSSNTAATNADCAWLEAQEDEEVGFPVKPQVPLRPMTYKAAFDLSFFLKEKGGLDGLIYSKKRQEILDLWVYNTQGFFPDWQNYTPGPGVRYPLTFGWCFKLVPVDPREVEEANKGENNCLLHPMNLHGMDDPEREVLVWRFDSRLAFHHMAREKHP EYYKNCII. 2-VALENT MMOSAIC ENV GP140 SEQUENCES (CLEAVAGE/FUSION-DEFECTIVE)MOSAIC ENV1 GP140 (AA SEQUENCE) SEQ ID NO: 9MRVTGIRKNYQHLWRWGTMLLGILMICSAAGKLWVTVYYGVPVWKEATTTLFCASDAKAYDTEVHNVWATHACVPTDPNPQEVVLENVTENFNMWKNNMVEQMHEDIISLWDQSLKPCVKLTPLCVTLNCTDDVRNVTNNATNTNSSWGEPMEKGEIKNCSFNITTSIRNKVQKQYALFYKLDVVPIDNDSNNTNYRLISCNTSVITQACPKVSFEPIPIHYCAPAGFAILKCNDKKFNGTGPCTNVSTVQCTHGIRPVVSTQLLLNGSLAEEEVVIRSENFTNNAKTIMVQLNVSVEINCTRPNNNTRKSIHIGPGRAFYTAGDIIGDIRQAHCNISRANWNNTLRQIVEKLGKQFGNNKTIVFNHSSGGDPEIVMHSFNCGGEFFYCNSTKLFNSTWTWNNSTWNNTKRSNDTEEHITLPCRIKQIINMWQEVGKAMYAPPIRGQIRCSSNITGLLLTRDGGNDTSGTEIFRPGGGDMRDNWRSELYKYKVVKIEPLGVAPTKAKRRVVQSEKSAVGIGAVFLGFLGAAGSTMGAASMTLTVQARLLLSGIVQQQNNLLRAIEAQQHLLQLTVWGIKQLQARVLAVERYLKDQQLLGIWGCSGKLICTTTVPWNASWSNKSLDKIWNNMTWMEWEREINNYTSLIYTLIEESQNQQEKNEQELLELDKWASLWNWFDISNWLW MOSAIC ENV2 GP140 (AA SEQUENCE)SEQ ID NO: 10 MRVRGIQRNWPQWWIWGILGFWMIIICRVMGNLWVTVYYGVPVWKEAKTTLFCASDAKAYEKEVHNVWATHACVPTDPNPQEMVLENVTENFNMWKNDMVDQMHEDIIRLWDQSLKPCVKLTPLCVTLECRNVRNVSSNGTYNIIHNETYKEMKNCSFNATTVVEDRKQKVHALFYRLDIVPLDENNSSEKSSENSSEYYRLINCNTSAITQACPKVSFDPIPIHYCAPAGYAILKCNNKTENGTGPCNNVSTVQCTHGIKPVVSTQLLLNGSLAEEEIIIRSENLTNNAKTIIVHLNETVNITCTRPNNNTRKSIRIGPGQTFYATGDIIGDIRQAHCNLSRDGWNKTLQGVKKKLAEHFPNKTINFTSSSGGDLEITTHSENCRGEFFYCNTSGLFNGTYMPNGTNSNSSSNITLPCRIKQIINMWQEVGRAMYAPPIAGNITCRSNITGLLLTRDGGSNNGVPNDTETFRPGGGDMRNNWRSELYKYKVVEVKPLGVAPTEAKRRVVESEKSAVGIGAVFLGILGAAGSTMGAASITLTVQARQLLSGIVQQQSNLLRAIEAQQHMLQLTVWGIKQLQTRVLAIERYLQDQQLLGLWGCSGKLICTTAVPWNTSWSNKSQTDIWDNMTWMQWDKEIGNYTGEIYRLLEESQNQQEKNEKDLLALDSWKNLWNWFDITNWLW MOS3 ENV GP140 (AA SEQUENCE) 678 AASEQ ID NO: 11 MRVKGIRKNYQHLWKWGTMLLGMLMICSAAEQLWVTVYYGVPVWRDAETTLFCASDAKAYEREVHNIWATHACVPTDPNPQEIVLENVTEEFNMWKNDMVEQMHTDIISLWDESLKPCVKLAPLCVTLNCTNANLNCTNDNCNRTVDKMREEIKNCSFNMTTELRDKKQKVYALFYKLDIVPIEKNSSEYRLINCNTSTITQACPKVTFEPIPIHYCTPAGFAILKCKDKKFNGTGPCKNVSTVQCTHGIKPVISTQLLLNGSLAEGEIIIRSENITNNAKTIIVQLNESVVINCTRPGNNTRKSVRIGPGQAFYATGEIIGDIRQAYCNISRAKWNNTLKQIVTKLKEQFKNKTIVFNQSSGGDPEITTHSFNCGGEFFYCNTTQLFNSTWNSNSTWNDTTGSVTEGNDTITLPCRIKQIVNMWQRVGQAMYAPPIEGNITCKSNITGLLLVRDGGNINRTNETFRPGGGNMKDNWRSELYKYKVVEIKPLGVAPTRAKRRVVESEKSAVGLGAVFLGFLGTAGSTMGAASLTLTVQARQVLSGIVQQQSNLLKAIEAQQHLLKLTVWGIKQLQARILAVERYLRDQQLLGIWGCSGKLICTTNVPWNSSWSNKSQEEIWNNMTWMQWDREISNYTDTIYRLLEDSQNQQEKNEQDLLALDKWASLWNWFSITNWLWIII. 2-VALENT M MOSAIC POL SEQUENCES (EXTENSIVELYINACTIVATED, PR-DELETED, 9 A INACTIVATIONMUTATIONS TO ELIMINATE CATALYTIC ACTIVITY) MOSAIC POL1 (AA SEQUENCE)SEQ ID NO: 12 MAPISPIETVPVKLKPGMDGPRVKQWPLTEEKIKALTAICEEMEKEGKITKIGPENPYNTPVFAIKKKDSTKWRKLVDFRELNKRTQDFWEVQLGIPHPAGLKKKKSVTVLAVGDAYFSVPLDEGFRKYTAFTIPSTNNETPGIRYQYNVLPQGWKGSPAIFQCSMTRILEPFRAKNPEIVIYQYMAALYVGSDLEIGQHRAKIEELREHLLKWGFTTPDKKHQKEPPFLWMGYELHPDKWTVQPIQLPEKDSWTVNDIQKLVGKLNWASQIYPGIKVRQLCKLLRGAKALTDIVPLTEEAELELAENREILKEPVHGVYYDPSKDLIAEIQKQGHDQWTYQIYQEPFKNLKTGKYAKMRTAHTNDVKQLTEAVQKIAMESIVIWGKTPKFRLPIQKETWETWWTDYWQATWIPEWEFVNTPPLVKLWYQLEKDPIAGVETFYVAGAANRETKLGKAGYVTDRGRQKIVSLTETTNQKTALQAIYLALQDSGSEVNIVTASQYALGIIQAQPDKSESELVNQIIEQLIKKERVYLSWVPAHKGIGGNEQVDKLVSSGIRKVLFLDGIDKAQEEHEKYHSNWRAMASDFNLPPVVAKEIVASCDQCQLKGEAMHGQVDCSPGIWQLACTHLEGKIILVAVHVASGYIEAEVIPAETGQETAYFILKLAGRWPVKVIHTANGSNFTSAAVKAACWWAGIQQEFGIPYNPQSQGVVASMNKELKKIIGQVRDQAEHLKTAVQMAVFIHNFKRKGGIGGYSAGERIIDIIATDIQTKELQKQIIKIQNFRVYYRDSRDPIWKGPAKLLWKGEGAVVIQDNSDIKVVPRRKVKIIKDYGKQMAGADCVAGRQDEDMOSAIC POL2 (AA SEQUENCE) SEQ ID NO: 13MAPISPIETVPVKLKPGMDGPKVKQWPLTEEKIKALVEICTEMEKEGKISKIGPENPYNTPIFAIKKKDSTKWRKLVDFRELNKRTQDFWEVQLGIPHPAGLKKKKSVTVLAVGDAYFSVPLDEDERKYTAFTIPSINNETPGIRYQYNVLPQGWKGSPAIFQSSMTKILEPFRKQNPDIVIYQYMAALYVGSDLEIGQHRTKIEELRQHLLRWGFTTPDKKHQKEPPFLWMGYELHPDKWTVQPIVLPEKDSWTVNDIQKLVGKLNWASQIYAGIKVKQLCKLLRGTKALTEVVPLTEEAELELAENREILKEPVHGVYYDPSKDLIAEIQKQGQGQWTYQIYQEPFKNLKTGKYARMRGAHTNDVKQLTEAVQKIATESIVIWGKTPKFKLPIQKETWEAWWTEYWQATWIPEWEFVNTPPLVKLWYQLEKEPIVGAETFYVAGAANRETKLGKAGYVTDRGRQKVVSLTDTTNQKTALQAIHLALQDSGLEVNIVTASQYALGIIQAQPDKSESELVSQIIEQLIKKEKVYLAWVPAHKGIGGNEQVDKLVSRGIRKVLFLDGIDKAQEEHEKYHSNWRAMASEFNLPPIVAKEIVASCDKCQLKGEAIHGQVDCSPGIWQLACTHLEGKVILVAVHVASGYIEAEVIPAETGQETAYFLLKLAGRWPVKTIHTANGSNFTSATVKAACWWAGIKQEFGIPYNPQSQGVVASINKELKKIIGQVRDQAEHLKTAVQMAVEIHNFKRKGGIGEYSAGERIVDIIASDIQTKELQKQITKIQNFRVYYRDSRDPLWKGPAKLLWKGEGAVVIQDNSDIKVVPRRKAKIIRDYGKQMAGDDCVASRQDEDMOS3 POL V3 (AA SEQUENCE) 851 AA SEQ ID NO: 14MAPISPIDTVPVTLKPGMDGPKIKQWPLTEEKIKALTEICTEMEKEGKISRIGPENPYNTPVFAIKKKNSTRWRKLVDFRELNKKTQDFWEVQLGIPHPAGLKKKRSVTVLAVGDAYFSVPLDKDFRKYTAFTIPSVNNETPGVRYQYNVLPQGWKGSPAIFQCSMTKILEPFRAQNPEIVIYQYVAALYVGSDLEIEQHRTKIEELRAHLLSWGFTTPDKKHQREPPFLWMGYELHPDRWTVQPIELPEKESWTVNDIQKLVGKLNWASQIYPGIKVKQLCRLLRGAKALTEVIPLTKEAELELAENREILREPVHGVYYDPSKDLVAEIQKQGQDQWTYQIYQEPYKNLKTGKYARKRSAHTNDVRQLTEAVQKIALESIVIWGKIPKFRLPIQRETWETWWTEYWQATWIPDWEFVNTPPLVKLWYQLEKEPIAGAETFYVAGASNRETKIGKAGYVTDKGRQKVVSLTETTNQKAALQAIQLALQDSGPEVNIVTASQYVLGIIQAQPDRSESELVNQIIEELIKKEKVYLSWVPAHKGIGGNEQVDKLVSAGIRKILFLDGIDKAQEEHERYHSNWRTMASDFNLPPIVAKEIVANCDKCQLKGEAMHGQVDCSPGMWQLACTHLEGKIIIVAVHVASGYMEAEVIPAETGQETAYYILKLAGRWPVKVVHTANGSNFTSTTVKAACWWANVTQEFGIPYNPQSQGVIASMNKELKKIIGQVREQAEHLKTAVQMAVLIHNFKRRGGIGGYSAGERIVDIIATDIQTRELQKQIIKIQNFRVYFRDSRDPVWKGPAKLLWKGEGAVVIQDNSEIKVVPRRKVKIIRDYGKQMAGDDCVAGRQDED QIV. 2-VALENT M MOSAIC GAG SEQUENCE MOS3 GAG (AA SEQUENCE) 508 AASEQ ID NO: 15 MGARASVLSGGKLDAWEKIRLRPGGKKKYKLKHIVWASRELDRFALNPGLLETAEGCQQIIEQLQPALQTGSEELKSLYNTVAVLYCVHQRIDVKDTKEALDKIEEIQNKSKQKTQQAAADTGSSSKVSQNYPIVQNAQGQMVHQALSPRTLNAWVKVVEEKGFNPEVIPMFSALAEGATPQDLNMMLNIVGGHQAAMQILKDTINEEAADWDRLHPVHAGPIPPGQMREPRGSDIAGTTSTPQEQIGWMTSNPPVPVGEIYKRWIIMGLNKIVRMYSPVSILDIKQGPKESFRDYVDRFFKVLRAEQATQEVKNWMTETLLIQNANPDCKSILRALGPGASLEEMMTACQGVGGPSHKARILAEAMSQANNTNIMMQRGNFKGQKRIKCFNCGKEGHLARNCRAPRKRGCWKCGREGHQMKDCNERQANFLGKIWPSSKGRPGNFPQSRPEPTAPLEPTAPPAEPTAPPAESFGFGEEITPSPKQEQKDREPLTSLKSL FGSDPLLQV. 2-VALENT M MOSAIC NEF SEQUENCES (POSITION 2G TO A TO DELETE MYRISTYLATION SITE MOS1 NEF (206 AA) SEQ ID NO: 16MAGKWSKSSVVGWPAIRERMRRAEPAADGVGAVSRDLEKHGAITSSNTAANNADCAWLEAQEEEEVGFPVRPQVPLRPMTYKGALDLSHFLKEKGGLEGLIYSQKRQDILDLWVYHTQGYFPDWQNYTPGPGIRYPLTFGWCFKLVPVEPEKIEEANEGENNSLLHPMSQHGMDDPEKEVLMWKFDSRLAFHHMARELHP EYYKDC MOS2 NEF(206 AA)-POSITION 2 G TO A TO DELETE MYRISTYLATION SITE SEQ ID NO: 17MAGKWSKSSIVGWPAVRERIRRAEPAAEGVGAASQDLDKYGALTSSNTAATNADCAWLEAQEDEEVGFPVKPQVPLRPMTYKAAFDLSFFLKEKGGLDGLIYSKKRQEILDLWVYNTQGFFPDWQNYTPGPGVRYPLTFGWCFKLVPVDPREVEEANKGENNCLLHPMNLHGMDDPEREVLVWRFDSRLAFHHMAREKHP EYYKNC MOS3 NEF(208 AA) SEQ ID NO: 18MAGKWSKRSVVGWPAVRERMRRTEPAAEGVGAVSQDLDKHGALTSSNTAHNNADCAWLQAQEEEEEVGFPVRPQVPVRPMTYKAAVDLSHFLKEKGGLEGLIHSQKRQEILDLWVYHTQGFFPDWHNYTPGPGTRFPLTFGWCYKLVPVDPKEVEEANEGENNCLLHPMSQHGMEDEDREVLKWKFDSSLARRHMARELH PEFYKDCLVI. 2-VALENT M MOSAIC GAGNEF FUSION SEQUENCESMOSAIC GAGNEF1 (AA SEQUENCE) SEQ ID NO: 19MGARASVLSGGELDRWEKIRLRPGGKKKYRLKHIVWASRELERFAVNPGLLETSEGCRQILGQLQPSLQTGSEELRSLYNTVATLYCVHQRIEIKDTKEALEKIEEEQNKSKKKAQQAAADTGNSSQVSQNYPIVQNIQGQMVHQAISPRTLNAWVKVVEEKAFSPEVIPMFSALSEGATPQDLNTMLNTVGGHQAAMQMLKETINEEAAEWDRVHPVHAGPIAPGQMREPRGSDIAGTTSTLQEQIGWMTNNPPIPVGEIYKRWIILGLNKIVRMYSPVSILDIRQGPKEPFRDYVDRFYKTLRAEQASQDVKNWMTETLLVQNANPDCKTILKALGPAATLEEMMTACQGVGGPGHKARVLAEAMSQVTNSATIMMQRGNFRNQRKTVKCFNCGKEGHIAKNCRAPRKKGCWKCGKEGHQMKDCTERQANFLGKIWPSNKGRPGNFLQNRPEPTAPPEESFRFGEETTTPSQKQEPIDKEMYPLASLKSLFGNDPSSQAGKWSKSSVVGWPAIRERMRRAEPAADGVGAVSRDLEKHGAITSSNTAANNADCAWLEAQEEEEVGFPVRPQVPLRPMTYKGALDLSHFLKEKGGLEGLIYSQKRQDILDLWVYHTQGYFPDWQNYTPGPGIRYPLTFGWCFKLVPVEPEKIEEANEGENNSLLHPMSQHGMDDPEKEVLMWKFDSRLAFHHMARELHPE YYKDCMOSAIC GAGNEF2 (AA SEQUENCE) SEQ ID NO: 20MGARASILRGGKLDKWEKIRLRPGGKKHYMLKHLVWASRELERFALNPGLLETSEGCKQIIKQLQPALQTGTEELRSLFNTVATLYCVHAEIEVRDTKEALDKIEEEQNKSQQKTQQAKEADGKVSQNYPIVQNLQGQMVHQPISPRTLNAWVKVIEEKAFSPEVIPMFTALSEGATPQDLNTMLNTVGGHQAAMQMLKDTINEEAAEWDRLHPVHAGPVAPGQMREPRGSDIAGTTSNLQEQIAWMTSNPPIPVGDIYKRWIILGLNKIVRMYSPTSILDIKQGPKEPFRDYVDRFFKTLRAEQATQDVKNWMTDTLLVQNANPDCKTILRALGPGATLEEMMTACQGVGGPSHKARVLAEAMSQTNSTILMQRSNFKGSKRIVKCFNCGKEGHIARNCRAPRKKGCWKCGKEGHQMKDCTERQANFLGKIWPSHKGRPGNFLQSRPEPTAPPAESFRFEETTPAPKQEPKDREPLTSLRSLFGSDPLSQAGKWSKSSIVGWPAVRERIRRAEPAAEGVGAASQDLDKYGALTSSNTAATNADCAWLEAQEDEEVGFPVKPQVPLRPMTYKAAFDLSFFLKEKGGLDGLIYSKKRQEILDLWVYNTQGFFPDWQNYTPGPGVRYPLTFGWCFKLVPVDPREVEEANKGENNCLLHPMNLHGMDDPEREVLVWRFDSRLAFHHMAREKHPEYYKNCVII. 2-VALENT M MOSAIC GAGPOL FUSION SEQUENCES(VERSION 3; POL EXTENSIVELY INACTIVATED,PR-DELETED, 9 A INACTIVATION MUTATIONS TO ELIMINATE CATALYTIC ACTIVITY)MOSAIC GAGPOL1 V3 (AA SEQUENCE) SEQ ID NO: 21MGARASVLSGGELDRWEKIRLRPGGKKKYRLKHIVWASRELERFAVNPGLLETSEGCRQILGQLQPSLQTGSEELRSLYNTVATLYCVHQRIEIKDTKEALEKIEEEQNKSKKKAQQAAADTGNSSQVSQNYPIVQNIQGQMVHQAISPRTLNAWVKVVEEKAFSPEVIPMFSALSEGATPQDLNTMLNTVGGHQAAMQMLKETINEEAAEWDRVHPVHAGPIAPGQMREPRGSDIAGTTSTLQEQIGWMTNNPPIPVGEIYKRWIILGLNKIVRMYSPVSILDIRQGPKEPFRDYVDRFYKTLRAEQASQDVKNWMTETLLVQNANPDCKTILKALGPAATLEEMMTACQGVGGPGHKARVLAEAMSQVTNSATIMMQRGNFRNQRKTVKCFNCGKEGHIAKNCRAPRKKGCWKCGKEGHQMKDCTERQANFLGKIWPSNKGRPGNFLQNRPEPTAPPEESFRFGEETTTPSQKQEPIDKEMYPLASLKSLFGNDPSSQMAPISPIETVPVKLKPGMDGPRVKQWPLTEEKIKALTAICEEMEKEGKITKIGPENPYNTPVFAIKKKDSTKWRKLVDFRELNKRTQDFWEVQLGIPHPAGLKKKKSVTVLAVGDAYFSVPLDEGFRKYTAFTIPSTNNETPGIRYQYNVLPQGWKGSPAIFQCSMTRILEPFRAKNPEIVIYQYMAALYVGSDLEIGQHRAKIEELREHLLKWGFTTPDKKHQKEPPFLWMGYELHPDKWTVQPIQLPEKDSWTVNDIQKLVGKLNWASQIYPGIKVRQLCKLLRGAKALTDIVPLTEEAELELAENREILKEPVHGVYYDPSKDLIAEIQKQGHDQWTYQTYQEPFKNLKTGKYAKMRTAHTNDVKQLTEAVQKIAMESIVIWGKTPKFRLPIQKETWETWWTDYWQATWIPEWEFVNTPPLVKLWYQLEKDPIAGVETFYVAGAANRETKLGKAGYVTDRGRQKIVSLTETTNQKTALQAIYLALQDSGSEVNIVTASQYALGIIQAQPDKSESELVNQIIEQLIKKERVYLSWVPAHKGIGGNEQVDKLVSSGIRKVLFLDGIDKAQEEHEKYHSNWRAMASDFNLPPVVAKEIVASCDQCQLKGEAMHGQVDCSPGIWQLACTHLEGKIILVAVHVASGYIEAEVIPAETGQETAYFILKLAGRWPVKVIHTANGSNFTSAAVKAACWWAGIQQEFGIPYNPQSQGVVASMNKELKKIIGQVRDQAEHLKTAVQMAVFIHNFKRKGGIGGYSAGERIIDIIATDIQTKELQKQIIKIQNFRVYYRDSRDPIWKGPAKLLWKGEGAVVIQDNSDIKVVPRRKVKIIKDYGKQMAGADCVAGRQDEDMOSAIC GAG POL2 V3 (AA SEQUENCE) SEQ ID NO: 22MGARASILRGGKLDKWEKIRLRPGGKKHYMLKHLVWASRELERFALNPGLLETSEGCKQIIKQLQPALQTGTEELRSLFNTVATLYCVHAEIEVRDTKEALDKIEEEQNKSQQKTQQAKEADGKVSQNYPIVQNLQGQMVHQPISPRTLNAWVKVIEEKAFSPEVIPMFTALSEGATPQDLNTMLNTVGGHQAAMQMLKDTINEEAAEWDRLHPVHAGPVAPGQMREPRGSDIAGTTSNLQEQIAWMTSNPPIPVGDIYKRWIILGLNKIVRMYSPTSILDIKQGPKEPFRDYVDRFFKTLRAEQATQDVKNWMTDTLLVQNANPDCKTILRALGPGATLEEMMTACQGVGGPSHKARVLAEAMSQTNSTILMQRSNFKGSKRIVKCFNCGKEGHIARNCRAPRKKGCWKCGKEGHQMKDCTERQANFLGKIWPSHKGRPGNFLQSRPEPTAPPAESFRFEETTPAPKQEPKDREPLTSLRSLFGSDPLSQMAPISPIETVPVKLKPGMDGPKVKQWPLTEEKIKALVEICTEMEKEGKISKIGPENPYNTPIFAIKKKDSTKWRKLVDFRELNKRTQDFWEVQLGIPHPAGLKKKKSVTVLAVGDAYFSVPLDEDFRKYTAFTIPSINNETPGIRYQYNVLPQGWKGSPAIFQSSMTKILEPFRKQNPDIVIYQYMAALYVGSDLEIGQHRTKIEELRQHLLRWGFTTPDKKHQKEPPFLWMGYELHPDKWTVQPIVLPEKDSWTVNDIQKLVGKLNWASQIYAGIKVKQLCKLLRGTKALTEVVPLTEEAELELAENREILKEPVHGVYYDPSKDLIAEIQKQGQGQWTYQIYQEPFKNLKTGKYARMRGAHTNDVKQLTEAVQKIATESIVIWGKTPKFKLPIQKETWEAWWTEYWQATWIPEWEFVNTPPLVKLWYQLEKEPIVGAETFYVAGAANRETKLGKAGYVTDRGRQKVVSLTDTTNQKTALQAIHLALQDSGLEVNIVTASQYALGIIQAQPDKSESELVSQIIEQLIKKEKVYLAWVPAHKGIGGNEQVDKLVSRGIRKVLFLDGIDKAQEEHEKYHSNWRAMASEFNLPPIVAKEIVASCDKCQLKGEAIHGQVDCSPGIWQLACTHLEGKVILVAVHVASGYIEAEVIPAETGQETAYFLLKLAGRWPVKTIHTANGSNFTSATVKAACWWAGIKQEFGIPYNPQSQGVVASINKELKKIIGQVRDQAEHLKTAVQMAVFIHNFKRKGGIGEYSAGERIVDIIASDIQTKELQKQITKIQNFRVYYRDSRDPLWKGPAKLLWKGEGAVVIQDNSDIKVVPRRKAKIIRDYGKQMAGDDCVASRQDED MOS3 GAG-POL V3 (AA SEQUENCES)1359 aa-GAG-POL FUSION WITH COMPLETE GAG AND MODIFIED POL SEQ ID NO: 23MGARASVLSGGKLDAWEKIRLRPGGKKKYKLKHIVWASRELDRFALNPGLLETAEGCQQIIEQLQPALQTGSEELKSLYNTVAVLYCVHQRIDVKDTKEALDKIEEIQNKSKQKTQQAAADTGSSSKVSQNYPIVQNAQGQMVHQALSPRTLNAWVKVVEEKGFNPEVIPMFSALAEGATPQDLNMMLNIVGGHQAAMQILKDTINEEAADWDRLHPVHAGPIPPGQMREPRGSDIAGTTSTPQEQIGWMTSNPPVPVGEIYKRWIIMGLNKIVRMYSPVSILDIKQGPKESFRDYVDRETKVLRAEQATQEVKNWMTETLLIQNANPDCKSILRALGPGASLEEMMTACQGVGGPSHKARILAEAMSQANNTNIMMQRGNFKGQKRIKCFNCGKEGHLARNCRAPRKRGCWKCGREGHQMKDCNERQANFLGKIWPSSKGRPGNFPQSRPEPTAPLEPTAPPAEPTAPPAESFGFGEEITPSPKQEQKDREPLTSLKSLFGSDPLLQMAPISPIDTVPVTLKPGMDGPKIKQWPLTEEKIKALTEICTEMEKEGKISRIGPENPYNTPVFAIKKKNSTRWRKLVDFRELNKKTQDFWEVQLGIPHPAGLKKKRSVTVLAVGDAYFSVPLDKDFRKYTAFTIPSVNNETPGVRYQYNVLPQGWKGSPAIFQCSMTKILEPFRAQNPEIVIYQYVAALYVGSDLEIEQHRTKIEELRAHLLSWGFTTPDKKHQREPPFLWMGYELHPDRWTVQPIELPEKESWTVNDIQKLVGKLNWASQIYPGIKVKQLCRLLRGAKALTEVIPLTKEAELELAENREILREPVHGVYYDPSKDLVAEIQKQGQDQWTYQIYQEPYKNLKTGKYARKRSAHTNDVRQLTEAVQKIALESIVIWGKIPKFRLPIQRETWETWWTEYWQATWIPDWEFVNTPPLVKLWYQLEKEPIAGAETFYVAGASNRETKIGKAGYVTDKGRQKVVSLTETTNQKAALQAIQLALQDSGPEVNIVTASQYVLGIIQAQPDRSESELVNQIIEELIKKEKVYLSWVPAHKGIGGNEQVDKLVSAGIRKILFLDGIDKAQEEHERYHSNWRTMASDFNLPPIVAKEIVANCDKCQLKGEAMHGQVDCSPGMWQLACTHLEGKIIIVAVHVASGYMEAEVIPAETGQETAYYILKLAGRWPVKVVHTANGSNFTSTTVKAACWWANVTQEFGIPYNPQSQGVIASMNKELKKIIGQVREQAEHLKTAVQMAVLIHNFKRRGGIGGYSAGERIVDIIATDIQTRELQKQIIKIQNFRVYFRDSRDPVWKGPAKLLWKGEGAVVIQDNSEIKVVPRRKVKIIRDYGKQMAGDDC VAGRQDEDQVIII. 2-VALENT M MOSAIC GAGPOL FUSION SEQUENCES(VERSION 4; POL MINIMALLY INACTIVATED, COMPLETE PR-RT-IN)MOSAIC GAGPOL1 V4 (AA SEQUENCE) SEQ ID NO: 24MGARASVLSGGELDRWEKIRLRPGGKKKYRLKHIVWASRELERFAVNPGLLETSEGCRQILGQLQPSLQTGSEELRSLYNTVATLYCVHQRIEIKDTKEALEKIEEEQNKSKKKAQQAAADTGNSSQVSQNYPIVQNIQGQMVHQAISPRTLNAWVKVVEEKAFSPEVIPMFSALSEGATPQDLNTMLNTVGGHQAAMQMLKETINEEAAEWDRVHPVHAGPIAPGQMREPRGSDIAGTTSTLQEQIGWMTNNPPIPVGEIYKRWIILGLNKIVRMYSPVSILDIRQGPKEPERDYVDREYKTLRAEQASQDVKNWMTETLLVQNANPDCKTILKALGPAATLEEMMTACQGVGGPGHKARVLAEAMSQVTNSATIMMQRGNFRNQRKTVKCFNCGKEGHIAKNCRAPRKKGCWKCGKEGHQMKDCTERQANFLGKIWPSNKGRPGNFLQNRPEPTAPPEESFRFGEETTTPSQKQEPIDKEMYPLASLKSLFGNDPSSQRENLAFQQGEAREFPSEQTRANSPTSRELQVRGDNPHSEAGAERQGTLNFPQITLWQRPLVSIKVGGQIREALLATGADDTVLEDINLPGKWKPKMIGGIGGFIKVGQYDQILIEICGKKAIGTVLVGPTPVNIIGRNMLTQLGCTLNFPISPIETVPVKLKPGMDGPRVKQWPLTEEKIKALTAICEEMEKEGKITKIGPENPYNTPVFAIKKKDSTKWRKLVDFRELNKRTQDFWEVQLGIPHPAGLKKKKSVTVLDVGDAYFSVPLDEGFRKYTAFTIPSTNNETPGIRYQYNVLPQGWKGSPAIFQCSMTRILEPFRAKNPEIVIYQYMDHLYVGSDLEIGQHRAKIEELREHLLKWGFTTPDKKHQKEPPFLWMGYELHPDKWTVQPIQLPEKDSWTVNDIQKLVGKLNWASQIYPGIKVRQLCKLLRGAKALTDIVPLTEEAELELAENREILKEPVHGVYYDPSKDLIAEIQKQGHDQWTYQIYQEPFKNLKTGKYAKMRTAHTNDVKQLTEAVQKIAMESIVIWGKTPKERLPIQKETWETWWTDYWQATWIPEWEEVNTPPLVKLWYQLEKDPIAGVETFYVDGAANRETKLGKAGYVTDRGRQKIVSLTETTNQKTELQAIYLALQDSGSEVNIVTDSQYALGIIQAQPDKSESELVNQIIEQLIKKERVYLSWVPAHKGIGGNEQVDKLVSSGIRKVLFLDGIDKAQEEHEKYHSNWRAMASDFNLPPVVAKEIVASCDQCQLKGEAMHGQVDCSPGIWQLACTHLEGKIILVAVHVASGYIEAEVIPAETGQETAYFILKLAGRWPVKVIHTDNGSNFTSAAVKAACWWAGIQQEFGIPYNPQSQGVVESMNKELKKIIGQVRDQAEHLKTAVQMAVFIHNFKRKGGIGGYSAGERIIDIIATDIQTKELQKQIIKIQNFRVYYRDSRDPIWKGPAKLLWKGEGAVVIQDNSDIKVVPRRKVKIIKDYGKQMAGADCVAGRQDEDMOSAIC GAGPOL2 V4 (AA SEQUENCE) SEQ ID NO: 25MGARASILRGGKLDKWEKIRLRPGGKKHYMLKHLVWASRELERFALNPGLLETSEGCKQIIKQLQPALQTGTEELRSLFNTVATLYCVHAEIEVRDTKEALDKIEEEQNKSQQKTQQAKEADGKVSQNYPIVQNLQGQMVHQPISPRTLNAWVKVIEEKAFSPEVIPMFTALSEGATPQDLNTMLNTVGGHQAAMQMLKDTINEEAAEWDRLHPVHAGPVAPGQMREPRGSDIAGTTSNLQEQIAWMTSNPPIPVGDIYKRWIILGLNKIVRMYSPTSILDIKQGPKEPFRDYVDRFFKTLRAEQATQDVKNWMTDTLLVQNANPDCKTILRALGPGATLEEMMTACQGVGGPSHKARVLAEAMSQTNSTILMQRSNFKGSKRIVKCFNCGKEGHIARNCRAPRKKGCWKCGKEGHQMKDCTERQANFLGKIWPSHKGRPGNFLQSRPEPTAPPAESFRFEETTPAPKQEPKDREPLTSLRSLFGSDPLSQRENLAFPQGKAREFSSEQTRANSPTRRELQVWGRDNNSLSEAGADRQGTVSFSFPQITLWQRPLVTIKIGGQLKEALLATGADDTVLEEMNLPGRWKPKMIGGIGGFIKVGQYDQIPIEICGHKAIGTVLVGPTPVNIIGRNLLTQIGCTLNFPISPIETVPVKLKPGMDGPKVKQWPLTEEKIKALVEICTEMEKEGKISKIGPENPYNTPIFAIKKKDSTKWRKLVDFRELNKRTQDFWEVQLGIPHPAGLKKKKSVTVLDVGDAYESVPLDEDFRKYTAFTIPSINNETPGIRYQYNVLPQGWKGSPAIFQSSMTKILEPERKQNPDIVIYQYMDHLYVGSDLEIGQHRTKIEELRQHLLRWGFTTPDKKHQKEPPFLWMGYELHPDKWTVQPIVLPEKDSWTVNDIQKLVGKLNWASQIYAGIKVKQLCKLLRGTKALTEVVPLTEEAELELAENREILKEPVHGVYYDPSKDLIAEIQKQGQGQWTYQIYQEPFKNLKTGKYARMRGAHTNDVKQLTEAVQKIATESIVIWGKTPKFKLPIQKETWEAWWTEYWQATWIPEWEFVNTPPLVKLWYQLEKEPIVGAETFYVDGAANRETKLGKAGYVTDRGRQKVVSLTDTTNQKTELQAIHLALQDSGLEVNIVTDSQYALGIIQAQPDKSESELVSQIIEQLIKKEKVYLAWVPAHKGIGGNEQVDKLVSRGIRKVLFLDGIDKAQEEHEKYHSNWRAMASEFNLPPIVAKEIVASCDKCQLKGEAIHGQVDCSPGIWQLACTHLEGKVILVAVHVASGYIEAEVIPAETGQETAYFLLKLAGRWPVKTIHTDNGSNFTSATVKAACWWAGIKQEFGIPYNPQSQGVVESINKELKKIIGQVRDQAEHLKTAVQMAVFIHNFKRKGGIGEYSAGERIVDIIASDIQTKELQKQITKIQNFRVYYRDSRDPLWKGPAKLLWKGEGAVVIQDNSDIKVVPRRKAKIIRDYGKQMAGDDCVASRQDEDIX. 2-VALENT M MOSAIC GAGPOL FUSION SEQUENCES(VERSION 5; POL MINIMALLY INACTIVATED, PR-DELETED)MOSAIC GAGPOL1 V5 (AA SEQUENCE) SEQ ID NO: 26MGARASVLSGGELDRWEKIRLRPGGKKKYRLKHIVWASRELERFAVNPGLLETSEGCRQILGQLQPSLQTGSEELRSLYNTVATLYCVHQRIEIKDTKEALEKIEEEQNKSKKKAQQAAADTGNSSQVSQNYPIVQNIQGQMVHQAISPRTLNAWVKVVEEKAFSPEVIPMFSALSEGATPQDLNTMLNTVGGHQAAMQMLKETINEEAAEWDRVHPVHAGFIAPGQMREPRGSDIAGTTSTLQEQIGWMTNNPPIPVGEIYKRWIILGLNKIVRMYSPVSILDIRQGPKEPFRDYVDRFYKTLRAEQASQDVKNWMTETLLVQNANPDCKTILKALGPAATLEEMMTACQGVGGPGHKARVLAEAMSQVTNSATIMMQRGNFRNQRKTVKCFNCGKEGHIAKNCRAPRKKGCWKCGKEGHQMKDCTERQANFLGKIWPSNKGRPGNFLQNRPEPTAPPEESERFGEETTIPSQKQEPIDKEMYPLASLKSLFGNDPSSQMAPISPIETVPVKLKPGMDGPRVKQWPLTEEKIKALTAICEEMEKEGKITKIGPENPYNTPVFAIKKKDSTKWRKLVDFRELNKRTQDFWEVQLGIPHPAGLKKKKSVTVLDVGDAYFSVPLDEGFRKYTAFTIPSTNNETPGIRYQYNVLPQGWKGSPAIFQCSMTRILEPFRAKNPEIVIYQYMDHLYVGSDLEIGQHRAKIEELREHLLKWGETTPDKKHQKEPPFLWMGYELHPDKWTVQPIQLPEKDSWTVNDIQKLVGKLNWASQIYPGIKVRQLCKLLRGAKALTDIVPLTEEAELELAENREILKEPVHGVYYDPSKDLIAEIQKQGHDQWTYQIYQEPFKNLKTGKYAKMRTAHTNDVKQLTEAVQKIAMESIVIWGKTPKERLPIQKETWETWWTDYWQATWIPEWEEVNTPPLVKLWYQLEKDPIAGVETFYVDGAANRETKLGKAGYVTDRGRQKIVSLTETTNQKTELQAIYLALQDSGSEVNIVTDSQYALGIIQAQPDKSESELVNQIIEQLIKKERVYLSWVPAHKGIGGNEQVDKLVSSGIRKVLELDGIDKAQEEHEKYHSNWRAMASDFNLPPVVAKEIVASCDQCQLKGEAMHGQVDCSPGIWQLACTHLEGKIILVAVHVASGYIEAEVIPAETGQETAYFILKLAGRWPVKVIHTDNGSNFTSAAVKAACWWAGIQQEFGIPYNPQSQGVVESMNKELKKIIGQVRDQAEHLKTAVQMAVFIHNFKRKGGIGGYSAGERIIDIIATDIQTKELQKQIIKIQNFRVYYRDSRDPIWKGPAKLLWKGEGAVVIQDNSDIKVVPRRKVKIIKDYGKQMAGADCVAGRQDEDMOSAIC GAGPOL2 V5 (AA SEQUENCE) SEQ ID NO: 27MGARASILRGGKLDKWEKIRLRPGGKKHYMLKHLVWASRELERFALNPGLLETSEGCKQIIKQLQPALQIGTEELRSLFNIVATLYCVHAEIEVRDTKEALDKIEEEQNKSQQKTQQAKEADGKVSQNYPIVQNLQGQMVHQPISPRILNAWVKVIEEKAFSPEVIPMFTALSEGATPQDLNTMLNTVGGHQAAMQMLKDTINEEAAEWDRLHPVHAGPVAPGQMREPRGSDIAGITSNLQEQIAWMTSNPPIPVGDIYKRWIILGLNKIVRMYSPTSILDIKQGPKEPFRDYVDRFFKTLRAEQATQDVKNWMTDILLVQNANPDCKTILRALGPGATLEEMMTACQGVGGPSHKARVLAEAMSQINSTILMQRSNFKGSKRIVKCFNCGKEGHIARNCRAPRKKGCWKCGKEGHQMKDCTERQANFLGKIWPSHKGRPGNFLQSRPEPTAPPAESFRFEETTPAPKQEPKDREPLISLRSLFGSDPLSQMAPISPIETVPVKLKPGMDGPKVKQWPLTEEKIKALVEICTEMEKEGKISKIGPENPYNTPIFAIKKKDSTKWRKLVDFRELNKRTINFWEVQLGIPHPAGLKKKKSVIVLDVGDAYFSVPLDEDFRKYTAFTIPSINNETPGIRYQYNVLPQGWKGSPAIFQSSMTKILEPFRKQNPDIVIYQYMDHLYVGSDLEIGQHRTKIEELRQHLLRWGFTTPDKKHQKEPPFLWMGYELHPDKWTVQPIVLPEKDSWTVNDIQKLVGKLNWASQIYAGIKVKQLCKLLRGTKALTEVVPLTEEAELELAENREILKEPVHGVYYDPSKDLIAEIQKQGQGQWTYQIYQEPFKNLKTGKYARMRGAHTNDVKQLTEAVQKIATESIVIWGKTPKFKLPIQKETWEAWWTEYWQATWIPEWEFVNTPPLVKLWYQLEKEPIVGAEIFYVDGAANRETKLGKAGYVTDRGRQKVVSLIDTTNQKTELQAIHLALQDSGLEVNIVIDSQYALGIIQAQPDKSESELVSQIIEQLIKKEKVYLAWVPAHKGIGGNEQVDKLVSRGIRKVLFLDGIDKAQEEHEKYHSNWRAMASEFNLPPIVAKEIVASCDKCQLKGEAIHGQVDCSPGIWQLACTHLEGKVILVAVHVASGYIEAEVIPAETGQETAYFLLKLAGRWPVKTIHTDNGSNFTSATVKAACWWAGIKQEFGIPYNPQSQGVVESINKELKKIIGQVRDQAEHLKIAVQMAVFIHNFKRKGGIGEYSAGERIVDIIASDIQTKELQKQIIKIQNFRVYYRDSRDPLWKGPAKLLWKGEGAVVIQDNSDIKVVPRRKAKIIRDYGKQMAGDDCVASRQDEDX. 2-VALENT M MOSAIC GAGPOLNEF FUSIONSEQUENCES (POL EXTENSIVELY INACTIVATED, PR-DELETED)MOSAIC GAGPOLNEF1 (AA SEQUENCE) SEQ ID NO: 28MGARASVLSGGELDRWEKIRLRPGGKKKYRLKHIVWASRELERFAVNPGLLETSEGCRQILGQLQPSLQTGSEELRSLYNTVATLYCVHQRIEIKDTKEALEKIEEEQNKSKKKAQQAAADIGNSSQVSQNYPIVQNIQGQMVHQAISPRILNAWVKVVEEKAFSPEVIPMFSALSEGATPQDLNTMLNTVGGHQAAMQMLKETINEEAAEWDRVHPVHAGPIAPGQMREPRGSDIAGITSTLQEQIGWMTNNPPIPVGEIYKRWIILGLNKIVRMYSPVSILDIRQGPKEPFRDYVDREYKTLRAEQASQDVKNWMTETLLVQNANPDCKTILKALGPAATLEEMMTACQGVGGPGHKARVLAEAMSQVTNSATIMMQRGNFRNQRKTVKCFNCGKEGHIAKNCRAPRKKGCWKCGKEGHQMKDCTERQANFLGKIWPSNKGRPGNFLQNRPEPTAPPEESFRFGEETTTPSQKQEPIDKEMYPLASLKSLFGNDPSSQMAPISPIETVPVKLKPGMDGPRVKQWPLTEEKIKALTAICEEMEKEGKITKIGPENPYNTPVFAIKKKDSTKWRKLVDFRELNKRTQDFWEVQLGIPHPAGLKKKKSVTVLAVGDAYFSVPLDEGFRKYTAFTIPSTNNETPGIRYQYNVLPQGWKGSPAIFQCSMTRILEPFRAKNPEIVIYQYMAALYVGSDLEIGQHRAKIEELREHLLKWGETTPDKKHQKEPPFLWMGYELHPDKWTVQPIQLPEKDSWTVNDIQKLVGKLNWASQIYPGIKVRQLCKLLRGAKALTDIVPLTEEAELELAENREILKEPVHGVYYDPSKDLIAEIQKQGHDQWTYQIYQEPFKNLKTGKYAKMRTAHTNDVKQLTEAVQKIAMESIVIWGKTPKFRLPIQKETWETWWTDYWQATWIPEWEEVNTPPLVKLWYQLEKDPIAGVETFYVAGAANRETKLGKAGYVTDRGRQKIVSLTETTNQKTALQAIYLALQDSGSEVNIVTASQYALGIIQAQPKSESELVNQIIEQLIKKERVYLSWVPAHKGIGGDNEQVDKLVSSGIRKVLFLDGIDKAQEEHEKYHSNWRAMASDFNLPPVVAKEIVASCDQCQLKGEAMHGQVDCSPGIWQLACTHLEGKIILVAVHVASGYIEAEVIPAETGQETAYFILKLAGRWPVKVIHTANGSNFTSAAVKAACWWAGIQQEFGIPYNPQSQGVVASMNKELKKIIGQVRDQAEHLKTAVQMAVFIHNFKRKGGIGGYSAGERIIDIIATDIQTKELQKQIIKIQNFRVYYRDSRDPIWKGPAKLLWKGEGAVVIQDNSDIKVVPRRKVKIIKDYGKQMAGADCVAGRQDEDMAGKWSKSSVVGWPAIRERMRRAEPAADGVGAVSRDLEKHGAITSSNTAANNADCAWLEAQEEEEVGFPVRPQVPLRPMTYKGALDLSHFLKEKGGLEGLIYSQKRQDILDLWVYHTQGYFPDWQNYTPGPGIRYPLTEGWCFKLVPVEPEKIEEANEGENNSLLHPMSQHGMDDPEKEVLMWKFDSRLAFHHMARELHP EYYKDCMOSAIC GAGPOLNEF2 (AA SEQUENCE) SEQ ID NO: 29MGARASILRGGKLDKWEKIRLRPGGKKHYMLKHLVWASRELERFALNPGLLETSEGCKQIIKQLQPALQTGTEELRSLENTVATLYCVHAEIEVRDTKEALDKIEEEQNKSQQKTQQAKEADGKVSQNYPIVQNLQGQMVHQPISPRTLNAWVKVIEEKAFSPEVIPMFTALSEGATPQDLNTMLNTVGGHQAAMQMLKDTINEEAAEWDRLHPVHAGPVAPGQMREPRGSDIAGTTSNLQEQIAWMTSNPPIPVGDIYKRWIILGLNKIVRMYSPTSILDIKQGPKEPERDYVDRFFKTLRAEQATQDVKNWMTDTLLVQNANPDCKTILRALGPGATLEEMMTACQGVGGPSHKARVLAEAMSQTNSTILMQRSNFKGSKRIVKCFNCGKEGHIARNCRAPRKKGCWKCGKEGHQMKDCTERQANFLGKIWPSHKGRPGNFLQSRPEPTAPPAESFRFEETTPAPKQEPKDREPLTSLRSLFGSDPLSQMAPISPIETVPVKLKPGMDGPKVKQWPLTEEKIKALVEICTEMEKEGKISKIGPENPYNTPIFAIKKKDSTKWRKLVDFRELNKRTQDFWEVQLGIPHPAGLKKKKSVTVLAVGDAYFSVPLDEDFRKYTAFTIPSINNETPGIRYQYNVLPQGWKGSPAIFQSSMTKILEPFRKQNPDIVIYQYMAALYVGSDLEIGQHRTKIEELRQHLLRWGFTTPDKKHQKEPPFLWMGYELHPDKWTVQPIVLPEKDSWTVNDIQKLVGKLNWASQIYAGIKVKQLCKLLRGTKALTEVVPLTEEAELELAENREILKEPVHGVYYDPSKDLIAEIQKQGQGQWTYQIYQEPFKNLKTGKYARMRGAHTNDVKQLTEAVQKIATESIVIWGKTPKFKLPIQKETWEAWWTEYWQATWIPEWEFVNTPPLVKLWYQLEKEPIVGAETFYVAGAANRETKLGKAGYVTDRGRQKVVSLTDTTNQKTALQAIHLALQDSGLEVNIVTASQYALGIIQAQPDKSESELVSQIIEQLIKKEKVYLAWVPAHKGIGGNEQVDKLVSRGIRKVLFLDGIDKAQEEHEKYHSNWRAMASEFNLPPIVAKEIVASCDKCQLKGEATHGQVDCSPGIWQLACTHLEGKVILVAVHVASGYIEAEVIPAETGQETAYFLLKLAGRWPVKTIHTANGSNFTSATVKAACWWAGIKQEFGIPYNPQSQGVVASINKELKKIIGQVRDQAEHLKTAVQMAVFIHNFKRKGGIGEYSAGERIVDIIASDIQTKELQKQITKIQNFRVYYRDSRDPLWKGPAKLLWKGEGAVVIQDNSDIKVVPRRKAKIIRDYGKQMAGDDCVASRQDEDMAGKWSKSSIVGWPAVRERIRRAEPAAEGVGAASQDLDKYGALTSSNTAATNADCAWLEAQEDEEVGFPVKPQVPLRPMTYKAAFDLSFFLKEKGGLDGLIYSKKRQEILDLWVYNTQGFFPDWQNYTPGPGVRYPLTFGWCFKLVPVDPREVEEANKGENNCLLHPMNLHGMDDPEREVLVWRFDSRLAFHHMAREKHPEYYKNCXI. OPTIMAL CLADE C ENV GP160, GAG, POL, NEF SEQUENCES OPTIMAL CLADE C ENV GP160(SN90.90.SE364) (AA SEQUENCE) SEQ ID NO: 30MRVTGMLRNCQPWWIWGILGFWMLLIYNVGGNLWVTVYYGVPVWKEAKTTLFCASDAKAYEKEVHNVWATHACVPTDPNPQEMVLENVTEYFNMWKNDMVDQMHEDIISLWDQSLKPCVKLTPLCVTLNCRNVTTSNNATSNDNPNGEIKNCSFNITTELRDKRRNEYALFYRLDIVPLSGSKNSSNSSEYRLINCNTSAITQACPKVSFDPIPIHYCAPAGYAILKCNNKTFNGTGPCNNVSTVQCTHGIKPVVSTQLLLNGSLAEGEIIIRSENLTNNAKTIIVHLNESIEIVCARPNNNTRKSMRIGPGQTFYATGDIIGDIRQAHCNISGNWNATLEKVKGKLQEHFPGKNISFEPSSGGDLEITTHSFNCRGEFFYCDTSKLFNGTTHTANSSITIQCRIKQIINMWQGVGRAIYAPPIAGNITCKSNITGLLLTRDGGTLNNDTEKFRPGGGDMRDNWRSELYKYKVVEIKPLGIAPTKAKRRVVEREKRAVGIGAVFLGFLGAAGSTMGAASITLTVQARQLLSGIVQQQSNLLRAIEAQQHMLQLTVWGIKQLQTRVLAIERYLKDQQLLGIWGCSGKIICTTAVPWNTSWSNKSLEDIWDNMTWMQWDREINNYTSIIYSLLEESQNQQEKNEKDLLALDSWNNLWNWFNITKWLWYIKIFIMIVGGLIGLRIIFAVLSIVNRVRQGYSPLSFQTLIPNPRGPDRLGRIEEEGGEQDRDRSIRLVNGFLAIAWDDLRSLCLFSYRRLRDFILIVARAVELLIQRGWETLKYLGSL?QYWGLELKKSAISLLDTIAITVAEGTDRIIELVQRICRAISNIPRRIRQGFEAALQOPTIMAL CLADE C GAG (IN.70177) (AA SEQUENCE) SEQ ID NO: 31MGARASILRGGKLDKWEKIRLRPGGKKHYMLKHLVWASRELERFALNPGLLETSEGCKQILKQLQPALQTGTEELRSLYNTVATLYCVHAGIEVRDTKEALDKIEEEQNKGQQKTQQAKGADGKVSQNYRIVQNLQGQMVHQAISPRTLNAWVKVIEEKAFSPEVIPMFTALSEGATPQDLNTMLNTVGGHQAAMQMLKDTINEEAAEWDRLHPVHAGPIAPGQMREPRGSDIAGTTSTLQEQIAWMTNNPPVPVGDIYKRWIILGLNKIVRMYSPVSILDIKQGPKEPFRDYVDRFFKTLRAEQATQDVKNWMTDTLLVQNANPDCKTILRALGPGATLEEMMTACQGVGGPSHKARVLAEAMSQTGSTIMMQRSNFKGSKRIVKCFNCGKEGHIARNCRAPRKKGCWKCGKEGHQMKDCTERQANFLGKIWPSHKGRPGNFLQSRPEPTAPPAESFRFEETTPAPKQELKDREPLTSLKSLFGSDPLSQOPTIMAL CLADE C POL (ZA.04.04ZASK208B1) (AA SEQUENCE) SEQ ID NO: 32FFRENLAFQQGEAREFPSEQARANSPTSREFQVRGDNPCSEAGVKGQGTLNFPQITLWQRPLVSIKVGGQVKEALLDTGADDTVLEEINLPGKWKPKMIGGIGGFIKVRQYDQILIEICGKKAIGTVLVGPTPVNIIGRNMLTQLGCTLNFPISPIETVPVKLKPGMDGPKIKQWPLTEEKIKALMAICEEMEKEGKITKIGPENPYNTPIFAIKKKDSTKWRKLVDFRELNKRTQDFWEVQLGIPHPAGLKKKKSVTVLDVGDAYFSVPLDESFRKYTAFTIPSINNETPGIRYQYNVLPQGWKGSPAIFQSSMTKILEPFRAKNPEIVIYQYMDDLYVGSDLEIGQHRAKIEELREHLLRWGFTTPDKKHQKEPPFLWMGYELHPDKWTVQPIQLPEKDSWTVNDIQKLVGKLNWASQIYSGIKVRQLCKLLRGAKALTDIVPLTEEAELELAENREILKEPVHGVYYDPSKDLIAEIQKQGYDQWTYQIYQEPFKNLKTGKYAKMRTAHTNDVKQLTEAVQKIALESIVIWGKTPKFRLPIQKETWEIWWTDYWQATWIPEWEFVNTPPLVKLWYQLEKEPIAGAETFYVDGAANRETKIGKAGYVTDKGRQKIVTLTETTNQKTELQAIQLALQDSGSEVNIVTDSQYALGIIQAQPDKSESELVNQIIEQLINKERVYLSWVPAHKGIGGNEQVDKLVSSGIRKVLFLDGIDKAQEEHEKYHSNWRAMASEFNLPPVVAKEIVASCDKCQLKGEAIHGQVDCSPGIWQLDCTHLEGKVILVAVHVASGYMEAEVIPAETGQETAYYILKLAGRWPVKVIHTDNGSNFTSAAVKAACWWAGIQQEFGIPYNPQSQGVVESMNKELKKIIGQVRDQAEHLKTAVQMAVFIHNFKRKGGIGGYSAGERIIDIIATDIQTKELQKQIIKIQNFRVYYRDSRDPIWKGPAKLLWKGEGAVVIQDNSDIKVVPRRKVKIIKDYGKQMAGADCVAGRQDEDOPTIMAL CLADE CNE F (ZA00.1170MB) (AA SEQUENCE) SEQ ID NO: 33MGGKWSKSSIVGWPDVRERMRRTEPAAEGVGAASQDLDKYGALTSSNTTHNNADCAWLEAQEEGEVGFPVRPQVPLRPMTYKGAFDLSFFLKEKGGLDGLIYSKKRQEILDLWVYHTQGFFPDWQNYTPGPGVRYPLTFGWCFKLVPVDPREVEEANKGENNCLLHPMSLHGMEDEEREVLKWEFDSSLARRHLARELHP EYYKDCXII. OPTIMAL CLADE C ENV GP140 SEQUENCE (CLEAVAGE/FUSION-DEFECTIVE)OPTIMAL CLADE C ENV GP140 (SN90.90.SE364) (AA SEQUENCE) SEQ ID NO: 34MRVTGMLRNCQPWWIWGILGFWMLLIYNVGGNLWVTVYYGVPVWKEAKTTLFCASDAKAYEKEVHNVWATHACVPTDPNPQEMVLENVTEYFNMWKNDMVDQMHEDIISLWDQSLKPCVKLTPLCVTLNCRNVTTSNNATSNDNPNGEIKNCSFNITTELRDKRRNEYALFYRLDIVPLSGSKNSSNSSEYRLINCNTSAITQACPKVSFDPIPIHYCAPAGYAILKCNNKTFNGTGPCNNVSTVQCTHGIKPVVSTQLLLNGSLAEGEIIIRSENLTNNAKTIIVHLNESIEIVCARPNNNTRKSMRIGPGQTFYATGDIIGDIRQAHCNISGNWNATLEKVKGKLQEHFPGKNISFEPSSGGDLEITTHSFNCRGEFFYCDTSKLFNGTTHTANSSITIQCRIKQIINMWQGVGRAIYAPPIAGNITCKSNITGLLLTRDGGTLNNDTEKFRPGGGDMRDNWRSELYKYKVVEIKPLGIAPTKAKRRVVESEKSAVGIGAVFLGFLGAAGSTMGAASITLTVQARQLLSGIVQQQSNLLRAIEAQQHMLQLTVWGIKQLQTRVLAIERYLKDQQLLGIWGCSGKIICTTAVPWNTSWSNKSLEDIWDNMTWMQWDREINNYTSIIYSLLEESQNQQEKNEKDLLALDS WNNLWNWFNITKWLWXIII. OPTIMAL CLADE C POL SEQUENCE (EXTENSIVELY INACTIVATED, PR-DELETED)OPTIMAL CLADE C POL (ZA.04.04ZASK208B1) (AA SEQUENCE) SEQ ID NO: 35MAPISPIETVPVKLKPGMDGPKIKQWPLTEEKIKALMAICEEMEKEGKITKIGPENPYNTPIFAIKKKDSTKWRKLVDFRELNKRTQDFWEVQLGIPHPAGLKKKKSVTVLAVGDAYFSVPLDESFRKYTAFTIPSINNETPGIRYQYNVLPQGWKGSPAIFQSSMTKILEPFRAKNPEIVIYQYMAALYVGSDLEIGQHRAKIEELREHLLRWGFTTPDKKHQKEPPFLWMGYELHPDKWTVQPIQLPEKDSWTVNDIQKLVGKLNWASQIYSGIKVRQLCKLLRGAKALTDIVPLTEEAELELAENREILKEPVHGVYYDPSKDLIAEIQKQGYDQWTYQIYQEPFKNLKTGKYAKMRTAHTNDVKQLTEAVQKIALESIVIWGKTPKFRLPIQKETWEIWWTDYWQATWIPEWEFVNTPPLVKLWYQLEKEPIAGAETFYVAGAANRETKIGKAGYVTDKGRQKIVTLTETTNQKTALQAIQLALQDSGSEVNIVTASQYALGIIQAQPDKSESELVNQIIEQLINKERVYLSWVPAHKGIGGNEQVDKLVSSGIRKVLFLDGIDKAQEEHEKYHSNWRAMASEFNLPPVVAKEIVASCDKCQLKGEAIHGQVDCSPGIWQLACTHLEGKVILVAVHVASGYMEAEVIPAETGQETAYYILKLAGRWPVKVIHTANGSNFTSAAVKAACWWAGIQQEFGIPYNPQSQGVVASMNKELKKIIGQVRDQAEHLKTAVQMAVFIHNFKRKGGIGGYSAGERIIDIIATDIQTKELQKQIIKIQNFRVYYRDSRDPIWKGPAKLLWKGEGAVVIQDNSDIKVVPRRKVKIIKDYGKQMAGADCVAGRQDEDXIV. OPTIMAL CLADE C GAGNEF FUSION SEQUENCEOPTIMAL CLADE C GAGNEF (IN.70177-ZA00.1170MB) (AA SEQUENCE)SEQ ID NO: 36 MGARASILRGGKLDKWEKIRLRPGGKKHYMLKHLVWASRELERFALNPGLLETSEGCKQILKQLQPALQTGTEELRSLYNTVATLYCVHAGIEVRDTKEALDKIEEEQNKGQQKTQQAKGADGKVSQNYPIVQNLQGQMVHQAISPRTLNAWVKVIEEKAFSPEVIPMFTALSEGATPQDLNTMLNTVGGHQAAMQMLKDTINEEAAEWDRLHPVHAGPIAPGQMREPRGSDIAGTTSTLQEQIAWMTNNPPVPVGDIYKRWIILGLNKIVRMYSPVSILDIKQGPKEPFRDYVDRFFKTLRAEQATQDVKNWMTDTLLVQNANPDCKTILRALGPGATLEEMMTACQGVGGPSHKARVLAEAMSQTGSTIMMQRSNFKGSKRIVKCFNCGKEGHIARNCRAPRKKGCWKCGKEGHQMKDCTERQANFLGKIWPSHKGRPGNFLQSRPEPTAPPAESFRFEETTPAPKQELKDREPLTSLKSLFGSDPLSQAGKWSKSSIVGWPDVRERMRRTEPAAEGVGAASQDLDKYGALTSSNTTHNNADCAWLEAQEEGEVGFPVRPQVPLRPMTYKGAFDLSFFLKEKGGLDGLIYSKKRQEILDLWVYHTQGFFPDWQNYTPGPGVRYPLTFGWCFKLVPVDPREVEEANKGENNCLLHPMSLHGMEDEEREVLKWEFDSSLARRHLARELHPEYYKDC XV. CONSENSUS SEQUENCESM CONSENSUS ENV SEQ ID NO: 37MRVRGIQRNCQHLWRWGTLILGMLMICSAAENLWVTVYYGVPVWKEANTTLFCASDAKAYDTEVHNVWATHACVPTDPNPQEIVLENVTENFNMWKNNMVEQMHEDIISLWDQSLKPCVKLTPLCVTLNCTNVNVTNTTNNTEEKGEIKNCSFNITTEIRDKKQKVYALFYRLDVVPIDDNNNNSSNYRLINCNTSAITQACPKVSFEPIPIHYCAPAGFAILKCNDKKENGTGPCKNVSTVQCTHGIKPVVSTQLLLNGSLAEEEIIIRSENITNNAKTIIVQLNESVEINCTRPNNNTRKSIRIGPGQAFYATGDIIGDIRQAHCNISGTKWNKTLQQVAKKLREHFNNKTIIFKPSSGGDLEITTHSFNCRGEFFYCNTSGLFNSTWIGNGTKNNNNTNDTITLPCRIKQIINMWQGVGQAMYAPPIEGKITCKSNITGLLLTRDGGNNNTNETEIFRPGGGDMRDNWRSELYKYKVVKIEPLGVAPTKAKRRVVESEKSAVGIGAVFLGELGAAGSTMGAASITLTVQARQLLSGIVQQQSNLLRAIEAQQHLLQLTVWGIKQLQARVLAVERYLKDQQLLGIWGCSGKLICTTTVPWNSSWSNKSQDEIWDNMTWMEWEREINNYTDIIYSLIEESQNQQEKNEQELLALDKWASLWNWFDITNWLW M CONSENSUS GAG SEQ ID NO: 38MGARASVLSGGKLDAWEKIRLRPGGKKKYRLKHLVWASRELERFALNPGLLETSEGCKQIIGQLQPALQTGSEELRSLYNTVATLYCVHQRIEVKDTKEALEKIEEEQNKSQQKTQQAAADKGNSSKVSQNYPIVQNLQGQMVHQAISPRTLNAWVKVIEEKAFSPEVIPMFSALSEGATPQDLNTMLNTVGGHQAAMQMLKDTINEEAAEWDRLHPVHAGPIPPGQMREPRGSDIAGTTSTLQEQIAWMTSNPPIPVGEIYKRWIILGLNKIVRMYSPVSILDIRQGPKEPERDYVDRFEKTLRAEQATQDVKNWMTDTLLVQNANPDCKTILKALGPGATLEEMMTACQGVGGPGHKARVLAEAMSQVTNAAIMMQRGNFKGQRRIIKCFNCGKEGHIARNCRAPRKKGCWKCGKEGHQMKDCTERQANFLGKIWPSNKGRPGNFLQSRPEPTAPPAESFGFGEEITPSPKQEPKDKEPPLTSLKSLFGNDPLSQ M CONSENSUS POLSEQ ID NO: 39 MAPISPIETVPVKLKPGMDGPKVKQWPLTEEKIKALTEICTEMEKEGKISKIGPENPYNTPIFAIKKKIDSTKWRKLVDFRELNKRTUFWEVQLGIPHPAGLKKKKSVTVLDVGDAYFSVPLDEDFRKYTAFTIPSINNETPGIRYQYNVLPQGWKGSPAIFQSSMTKILEPFRTQNPEIVIYQYMDHLYVGSDLEIGQHRAKIEELREHLLRWGFTTPDKKHQKEPPFLWMGYELHPDKWTVQPIQLPEKDSWTVNDIQKLVGKLNWASQIYPGIKVKQLCKLLRGAKALTDIVPLTEEAELELAENREILKEPVHGVYYDPSKDLIAEIQKQGQDQWTYQIYQEPFKNLKTGKYAKMRSAHTNDVKQLTEAVQKIATESIVIWGKTPKFRLPIQKETWETWWTEYWQATWIPEWEFVNTPPLVKLWYQLEKEPIAGAETFYVDGAANRETKLGKAGYVTDRGRQKVVSLTETTNQKTELQAIHLALQDSGSEVNIVTDSQYALGIIQAQPDKSESELVNQIIEQLIKKEKVYLSWVPAHKGIGGNEQVDKLVSTGIRKVLFLDGIDKAQEEHEKYHSNWRAMASDFNLPPIVAKEIVASCDKCQLKGEAMHGQVDCSPGIWQLACTHLEGKIILVAVHVASGYIEAEVIPAETGQETAYFILKLAGRWPVKVIHTDNGSNFTSAAVKAACWWAGIQQEFGIPYNPQSQGVVESMNKELKKIIGQVRDQAEHLKTAVQMAVFIHNFKRKGGIGGYSAGERIIDIIATDIQTKELQKQITKIQNFRVYYRDSRDPIWKGPAKLLWKGEGAVVIQDNSDIKVVPRRKAKIIRDYGKQMAGDDCVAGRQDED

Other Embodiments

While the invention has been described in connection with specificembodiments thereof, it will be understood that it is capable of furthermodifications and this application is intended to cover any variations,uses, or adaptations of the invention following, in general, theprinciples of the invention and including such departures from thepresent disclosure that come within known or customary practice withinthe art to which the invention pertains and may be applied to theessential features hereinbefore set forth.

All publications and patent applications mentioned in this specificationare herein incorporated by reference to the same extent as if eachindependent publication or patent application was specifically andindividually indicated to be incorporated by reference in theirentirety.

What is claimed is:
 1. An immunogenic composition comprising at leasttwo distinct optimized viral polypeptides, wherein said optimized viralpolypeptides correspond to the same viral gene product, wherein theviral gene product is selected from the group consisting of gag, pol,and env, and wherein the at least two distinct optimized gagpolypeptides are selected from any one or more of the groups: (a) SEQ IDNO: 3 and 4, (b) SEQ ID NO: 3, and 15 (c) SEQ ID NO: 4 and 15; whereinthe at least two distinct optimized pol polypeptides are selected fromany one or more of the groups: (a) SEQ ID NO: 12 and 13, (b) SEQ ID NO:12 and 14, (c) SEQ ID NO: 13 and 14; and wherein the at least twodistinct optimized env polypeptides are selected from any one or more ofthe groups: (a) SEQ ID NO: 9 and 10, (b) SEQ ID NO: 9 and 11, (c) SEQ IDNO: 10 and
 11. 2. The immunogenic composition of claim 1, wherein saidmammal is a human.
 3. The immunogenic composition of claim 1, whereinsaid immunogenic composition elicits a cellular immune response againstsaid viral gene product.
 4. The immunogenic composition of claim 1,wherein said at least two optimized gag viral polypeptides comprise anamino acid sequence having the sequences set forth in each of SEQ IDNO:3 and
 4. 5. The immunogenic composition of claim 1, wherein said atleast two optimized pol viral polypeptides comprise an amino acidsequence having the sequences set forth in each of SEQ ID NO:12 and 13.6. The immunogenic composition of claim 4 further comprising at leasttwo optimized pol viral polypeptides, wherein said polypeptides comprisean amino acid sequence having the sequences set forth in each of SEQ IDNO:12 and
 13. 7. The immunogenic composition of claim 6 furthercomprising at least two optimized env viral polypeptides, wherein saidpolypeptides comprise an amino acid sequence having the sequences setforth in each of SEQ ID NO:9 and
 10. 8. The immunogenic composition ofclaim 1, wherein said at least two distinct optimized viral polypeptidesare encoded in a viral vector selected from the group consisting ofadenovirus serotype 26 (Ad26), adenovirus serotype 34 (Ad34), adenovirusserotype 35 (Ad35), adenovirus serotype 48 (Ad48), er adenovirusserotype 5 HVR48 (Ad5HVR48), poxvirus, and modified vaccinia virusAnkara (MVA).
 9. A method for inducing an HIV-1-specific immune responsein a mammal comprising administering to said mammal the immunogeniccomposition of claim
 1. 10. A method of manufacturing the immunogeniccomposition of claim 1 comprising synthesizing said optimized viralpolypeptides and combining said optimized viral polypeptides with apharmaceutically acceptable carrier.
 11. A kit comprising: a) theimmunogenic composition of claim 1; b) a pharmaceutically acceptablecarrier, excipient, or diluent; c) instructions for the use thereof;and, optionally, d) an adjuvant.
 12. The immunogenic composition ofclaim 8, wherein said vector is the Ad26 vector.
 13. The immunogeniccomposition of claim 8, wherein said vector is the MVA vector.
 14. Theimmunogenic composition of claim 1 admixed with a pharmaceuticallyacceptable carrier, excipient, or diluent.
 15. The immunogeniccomposition of claim 1, wherein said at least two optimized env viralpolypeptides comprise an amino acid sequence having the sequences setforth in each of SEQ ID NO:9 and
 11. 16. A method for inducing anHIV-1-specific immune response in a mammal comprising administering tosaid mammal the immunogenic composition of claim
 8. 17. The method ofclaim 16, wherein said vector is the Ad26 vector.
 18. The method ofclaim 16, wherein said vector is the MVA vector.
 19. A polypeptidecomprising the amino acid sequence of SEQ ID NO:
 9. 20. A method forinducing an HIV-1-specific immune response in a mammal comprisingadministering to said mammal the immunogenic composition of claim 19.21. The method of claim 20, wherein the mammal is a human.
 22. Themethod of claim 21, wherein said composition comprises apharmaceutically acceptable carrier, excipient, or diluent.
 23. Themethod of claim 16, wherein said mammal is a human.